U.S. patent application number 15/408563 was filed with the patent office on 2017-06-01 for nanostars and nanoconstructs for detection, imaging, and therapy.
The applicant listed for this patent is Duke University. Invention is credited to Andrew Fales, Christopher Khoury, Tuan Vo-Dinh, Hsiangkuo Yuan.
Application Number | 20170151331 15/408563 |
Document ID | / |
Family ID | 57909273 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170151331 |
Kind Code |
A1 |
Vo-Dinh; Tuan ; et
al. |
June 1, 2017 |
NANOSTARS AND NANOCONSTRUCTS FOR DETECTION, IMAGING, AND
THERAPY
Abstract
A polymer-free synthesis method is provided for preparation of
monodisperse nanostars. The nanostars can be used for treating and
imaging cells in in vivo or ex vivo. The modes of treatment include
use of a nanostar modified with a photo-activatable drug, which
drug is activated by the photo-response of the nanostar to
electromagnetic stimulation; use of a nanostar modified with a
thermally-activatable drug, which drug is activated by a thermal
response of the nanostar to electromagnetic stimulation; and the
thermal response of the nanostar itself to electromagnetic
stimulation, which can directly or indirectly cause the death of an
undesirable cell.
Inventors: |
Vo-Dinh; Tuan; (Chapel Hill,
NC) ; Yuan; Hsiangkuo; (Chalfont, PA) ; Fales;
Andrew; (Duham, NC) ; Khoury; Christopher;
(Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
57909273 |
Appl. No.: |
15/408563 |
Filed: |
January 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13971822 |
Aug 20, 2013 |
9561292 |
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15408563 |
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61691858 |
Aug 22, 2012 |
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61691004 |
Aug 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/1821 20130101;
A61K 41/0057 20130101; A61K 41/0052 20130101; G01N 33/582 20130101;
A61K 41/0071 20130101; A61K 41/0066 20130101 |
International
Class: |
A61K 41/00 20060101
A61K041/00; G01N 33/58 20060101 G01N033/58; A61K 49/18 20060101
A61K049/18 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with U.S. Government support under
the National Institutes of Health grant No's.: R01 EB006201, R01
ES014774 and T32 EB001040. The U.S. Government has certain rights
in the invention.
Claims
1. A plasmonics-active gold nanostar resulting from a process
comprising: reducing aqueous gold (Au.sup.3+) to solid gold (Au) in
an acidic solution; and mixing a silver salt compound with a weak
reducing agent into the solution under conditions such that the
plasmonics-active gold nanostars are produced.
2. The gold nanostar of claim 1, wherein the Au.sup.3+ comprises
tetrachloroauric acid (HAuCl.sub.4).
3. The gold nanostar of claim 1, wherein the Au.sup.3+ is reduced
to solid Au onto citrate-stabilized gold seeds.
4. The gold nanostar of claim 1, wherein the weak reducing agent
consists essentially of ascorbic acid.
5. The gold nanostar of claim 1, wherein the silver salt compound
consists essentially of silver nitrate (AgNO.sub.3).
6. The gold nanostar of claim 1, wherein the weak reducing agent
consists essentially of ascorbic acid, and wherein the ratio of the
ascorbic acid to the HAuCl.sub.4 ranges from about 1.5 to about
2.
7. The gold nanostar of claim 1, wherein a size of the nanostar is
less than about 100 nm.
8. The gold nanostar of claim 1, wherein the concentration of a
silver cation of the silver compound ranges from about 5 .mu.M to
about 30 .mu.M and a plasmon peak of the nanostar ranges from about
600 nm to about 1000 nm.
9. The gold nanostar of claim 3, wherein the citrate-stabilized
gold seeds comprise hollow gold nanoshells and the
plasmonics-active gold nanostars are hollow.
10. The gold nanostar of claim 3, wherein the citrate-stabilized
gold seeds comprise superparamagnetic particles coated with a layer
of gold, and the plasmonics-active gold nanostars are
superparamagnetic.
11. The gold nanostar of claim 10, wherein the superparamagnetic
particles comprise iron oxide (TO).
12. The gold nanostar of claim 1, wherein the nanostar further
comprises one or more of an optical label, a photosensitizer, and a
photoactivator.
13. The gold nanostar of claim 12, wherein the optical label
comprises one or more of a fluorescence label, fluorescein,
fluorescein isothiocyanate (FITC), thionine dyes, rhodamine,
crystal violet, Raman label, 3,3'-diethylthiatricarbocyanine iodide
(DTTC), absorbance label, positively-charged hydrophobic NIR dyes,
IR-780, IR-792, IR-797, IR-813, methylene blue hydrate (MB),
4-mercaptobenzoic acid (4-MBA), 5,5'-dithiobis-2-nitrobenzoic acid
(DTNB), and 4-aminothiophenol (4ATP).
14. The gold nanostar of claim 12, wherein the nanostar comprises
one or both of a layer surrounding the nanostar having within the
layer the one or more of the optical label, the photosensitizer,
and the photoactivator, and a protective overlayer surrounding the
layer.
15. A method for preparing plasmonics-active gold nanostars free of
polymer, the method comprising: reducing aqueous gold (Au.sup.3+)
to solid gold (Au) in an acidic solution; and mixing a silver salt
compound with a weak reducing agent into the solution under
conditions such that the plasmonics-active gold nanostars are
produced.
16. The method of claim 15, wherein the Au.sup.3+ comprises
tetrachloroauric acid (HAuCl.sub.4).
17. The method of claim 15, wherein the Au.sup.3+ is reduced to
solid Au onto citrate-stabilized gold seeds.
18. The method of claim 15, wherein the weak reducing agent
consists essentially of ascorbic acid.
19. The method of claim 15, wherein the silver salt compound
consists essentially of silver nitrate (AgNO.sub.3).
20. The method of claim 15, wherein the weak reducing agent
consists essentially of ascorbic acid, and wherein the ratio of the
ascorbic acid to the HAuCl.sub.4 ranges from about 1.5 to about
2.
21. The method of claim 15, wherein the concentration of a silver
cation of the silver compound ranges from about 5 .mu.M to about 30
.mu.M and a plasmon peak of the nanostar ranges from about 600 nm
to about 1000 nm.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/971,822 entitled "Nanostars and Nanoconstructs for
Detection, Imaging, and Therapy", filed on Aug. 20, 2013, which
further claims priority to U.S. Provisional Patent Application No.
61/691,858 filed Aug. 22, 2012, and U.S. Provisional Patent
Application No. 61/691,004 filed Aug. 20, 2012, the disclosures of
which are incorporated herein by reference in their entirety. This
application is related to U.S. patent application Ser. No.
13/888,226 filed May 6, 2013.
TECHNICAL FIELD
[0003] The present disclosure relates to metal nanostars.
Particularly, the present disclosure relates to methods for making
and using plasmonics-active metal nanostars to treat and detect
cells in vivo and ex vivo.
BACKGROUND
[0004] The development and fabrication of plasmonics-active
metallic nanostructures have been active areas of research for a
wide variety of applications. Plasmonics refers to the study of
enhanced electromagnetic properties of metallic nanostructures. The
term is derived from plasmons, the quanta associated with
longitudinal waves propagating in matter through the collective
motion of large numbers of electrons. According to classical
electromagnetic theory, molecules on or near metal nanostructures
experience enhanced fields relative to that of the incident
radiation. When a metallic nanostructured surface is irradiated by
an incident electromagnetic field (e.g., a laser beam), conduction
electrons are displaced into frequency oscillations equal to those
of the incident light. These oscillating electrons, called "surface
plasmons," produce a secondary electric field, which adds to the
incident field. The origin of plasmon resonances of metallic
nanoparticles is collective oscillations of the conduction band
electrons in the nanoparticles, which are called Localized Surface
Plasmons (LSPs). LSPs can be excited when light is incident on
metallic nanoparticles having a size much smaller than the
wavelength of the incident light. At a suitable wavelength,
resonant dipolar and multipolar modes can be excited in the
nanoparticles, which lead to a significant enhancement in absorbed
and scattered light and enhancement of electromagnetic fields
inside and near the particles. Hence, the LSPs can be detected as
resonance peaks in the absorption or scattering spectra of the
metallic nanoparticles. This condition yields intense localized
fields, which can interact with molecules in contact with or near
the metal surface. In an effect analogous to a "lightning rod"
effect, secondary fields can become concentrated at high curvature
points on the nanostructured metal surface.
[0005] Nanoparticles of noble metals such as gold and silver
resonantly scatter and absorb light in the visible and
near-infrared spectral region upon the excitation of their plasmon
and are therefore materials of choice for plasmon related devices.
Surface plasmons have been associated with important practical
applications in surface plasmon resonance (SPR), surface-enhanced
Raman scattering (SERS) and surface-enhanced luminescence, also
referred to as metal-enhanced luminescence. Such SERS technology
has been extensively investigated and a wide variety of
plasmonics-active SERS platforms developed for chemical sensing and
for bioanalysis and biosensing [T. Vo-Dinh, "Surface-Enhanced Raman
Spectroscopy Using Metallic Nanostructures," Trends in Anal. Chem.,
17, 557-582 (1998); T. Vo-Dinh, A. Dhawan, S. J. Norton, C. G.
Khoury, H-N. Wang, V. Misra, and M. Gerhold "Plasmonic
Nanoparticles and Nanowires: Design, Fabrication and Application in
Sensing", J. Phys. Chem. C, 114 (16), pp 7480-7488 (2010).
[0006] Photodynamic Therapy (PDT) is light-based treatment, which
involves treatment of diseases such as cancer using light action on
a special photoactive class of drugs, by photodynamic action in
vivo to destroy or modify tissue [Dougherty T. J. and Levy J. G.,
"Photodynamic Therapy and Clinical Applications", in Biomedical
Photonics Handbook, Vo-Dinh T., Ed., CRC Press, Boca Raton Fla.
(2003)]. PDT, which was originally developed for treatment of
various cancers, has now been used to include treatment of
pre-cancerous conditions, e.g. actinic keratoses, high-grade
dysplasia in Barrett's esophagus, and non-cancerous conditions,
e.g. various eye diseases, e.g. age related macular degeneration.
Photodynamic therapy (PDT) is approved for commercialization
worldwide both for various cancers (lung, esophagus) and for AMD.
The PDT process requires three elements: (1) photosensitizer), (2)
light that can excite the photosensitizer (Ps) and (3) endogenous
oxygen. The putative cytotoxic agent is singlet oxygen, an
electronically excited state of ground state triplet oxygen formed
according to Type II photochemical process.
[0007] Transition to the triplet state is important since the
triplet state has a relatively long lifetime (.mu.sec to seconds).
As a result photosensitizers that undergo efficient intersystem
crossing to the excited triplet state will have sufficient time for
collision with oxygen in order to produce singlet oxygen. The
energy difference between ground state and singlet oxygen is 94.2
kJ/mol and corresponds to a transition in the near-infrared (NIR)
at .about.1270 nm. Most PS photosensitizers in clinical use have
triplet quantum yields in the range of 40-60% with the singlet
oxygen yield being slightly lower. Competing processes include loss
of energy by deactivation to ground state by fluorescence or
internal conversion (loss of energy to the environment).
[0008] Many factors, including a high yield of singlet oxygen,
pharmacokinetics, pharmacodynamics, stability in vivo and
acceptable toxicity, play critical roles as well [Henderson B W,
Gollnick S O, "Mechanistic Principles of Photodynamic Therapy", in
Biomedical Photonics Handbook, Vo-Dinh T., Ed., CRC Press, Boca
Raton Fla. (2003)]. For example, it is desirable to have relatively
selective uptake in the tumor or other tissue being treated
relative to the normal tissue that necessarily will be exposed to
the exciting light as well. Pharmacodynamic issues such as the
subcellular localization of the photosensitizer may be important as
certain organelles appear to be more sensitive to PDT damage than
others (e.g. the mitochondria). Toxicity can become an issue if
high doses of photosensitizer are necessary in order to obtain a
complete response to treatment. An important mechanism associated
with PDT drug activity involves apoptosis in cells. Upon absorption
of light, the photosensitiser (Ps) initiates chemical reactions
that lead to the direct or indirect production of cytotoxic species
such as radicals and singlet oxygen. The reaction of the cytotoxic
species with subcellular organelles and macromolecules (proteins,
DNA, etc) lead to apoptosis and/or necrosis of the cells hosting
the PDT drug. The preferential accumulation of PDT drug molecules
in cancer cells combined with the localized delivery of light to
the tumor, results in the selective destruction of the cancerous
lesion. Compared to other traditional anticancer therapies, PDT
does not involve generalized destruction of healthy cells. In
addition to direct cell killing, PDT can also act on the
vasculature, reducing blood flow to the tumor causing its necrosis.
In particular cases it can be used as a less invasive alternative
to surgery.
[0009] There are several chemical species used for PDT including
porphyrin-based sensitizers. A purified hematoporphyrin derivative,
Photofrin.RTM., has received approval of the US Food and Drug
Administration. Porphyrins are generally used for tumors on or just
under the skin or on the lining of internal organs or cavities
because theses drug molecules absorb light shorter than 640 nm in
wavelength. For tumors occurring deep in tissue, second generation
sensitizers, which have absorbance in the NIR region, such as
porphyrin-based systems [R. K. Pandey, "Synthetic Strategies in
designing Porphyrin-Based Photosensitizers", in Biomedical
Photonics Handbook, Vo-Dinh T., Ed., CRC Press, Boca Raton Fla.
(2003)], chlorines, phthalocyanine, and naphthalocyanine have been
investigated.
[0010] Nanoparticle systems have gained wide attention due to their
potential in medicine, such as molecular imaging, immunization,
theranostics, and targeted delivery/therapy. Nanoparticles can be
fabricated as strong contrast agents for different imaging
modalities with superior signal-to-noise ratios than conventional
agents, or as therapeutic agents such as drug carriers,
radioenhancers, and photothermal transducers. Gold nanoparticles
(AuNPs), with their facile synthesis and biocompatibility, have
therefore been applied for a variety of therapeutics, especially in
cancer therapy.
[0011] These substrates consist of microplates, waveguides or
optical fibers having silver-coated dielectric nanoparticles or
isolated dielectric nanospheres coated with a silver nanolayer
producing nanocaps (i.e. half nanoshells), nanorods and nanostars.
These plasmonics substrate platforms have led to a wide variety of
analytical applications including sensitive detection of a variety
of chemicals of environmental, biological and medical significance,
including polycyclic aromatic compounds, organophosphorus
compounds, and compounds of biological interest such as DNA-adduct
biomarkers.
[0012] Gold nanostars (NS), with a high absorption-to-scattering
ratio in the NIR, efficiently transduce photon energy into heat for
hyperthermia therapy. To date, most phothermolysis studies utilize
laser irradiation higher than the maximal permissible exposure
(MPE) of skin by ANSI regulation. To make photothermolysis
applicable to real practice, one needs to enhance the photothermal
transduction efficiency. One way is to use a pulsed laser instead
of a continuous-wave laser, permitting efficient photothermal
conversion by allowing additional time for electron-phonon
relaxation. Previously, in vitro photothermolysis using NIR pulsed
laser reported irradiances of 1.6-48.6 W/cm.sup.2; which were
higher than the MPE of skin (e.g. 0.4 W/cm.sup.2 at 800 nm).
Insufficient intracellular particle delivery and low photothermal
transduction efficiency may be the main obstacles. Therefore, there
is a strong need to design a more efficient photothermal transducer
with optimized cellular uptake.
[0013] Recently, star-shaped AuNPs ("nanostars") have attracted
interest because their plasmon can be tuned to the NIR region, and
the structure contains multiple sharp tips that can greatly enhance
incident electromagnetic fields. Studies have shown that
NIR-absorbing nanorods, nanocages or nanoshells can be used as
contrast agents in optical imaging techniques such as optical
coherent tomography, two-photon luminescence (TPL) microscopy, and
photoacoustic imaging. Their large absorption cross-sections can
also effectively convert photon energy to heat during photothermal
therapy. Nanostars, which absorb in the NIR, have been hypothesized
to behave similarly. Nanostar-related bioapplications remain scarce
in spite of their potential, mostly due to the difficulty of
surface functionalization.
[0014] In 2003, Chen et al. [Chen S, Wang Z L, BaHato J, Foulger S
H, Carroll D L., J Am Chem Soc. 2003 Dec. 31; 125(52):16186-7]
first reported the synthesis of multipod gold nanoparticles from
silver plates in the presence of cetyltrimethylammonium bromide
(CTAB) and NaOH. Later, several seedless or seed-mediated synthesis
methods were employed using majorly poly(N-vinylpyrolidone) (PVP)
or CTAB as surfactant. Further use of nanostars has been limited by
(1) the potential toxicity of CTAB, (2) the difficulty of replacing
PVP or CTAB during biofunctionalization, and (3) induction of
aggregation following multiple washes. Previous experimental
studies have shown a red-shifting of the plasmon peak from
nanostars with longer or sharper branches. Several numerical
studies of their plasmonic properties have recently been reported.
Hao et al.'s [Hao F, Nehl C L, Hafner J H, Nordlander P. Nano Lett.
2007 March; 7(3):729-32] 2-D modeling of a single nanostar,
consisting of 5 unique branches, with finite difference time domain
(FDTD) method showed that nanostars plasmon results from the
hybridization of plasmon resonance of each branch; the plasmon peak
relative intensity depends on the polarization angle. Senthil et
al. [Senthil Kumar P, Pastoriza-Santos I, Rodriguez-Ganzalez B,
Garcia de Abajo F J, Liz-Marzan L M. Nanotechnology, 2008;
19(1):015606-12] also stated that the tip angle and radius, but not
the number of branches, are the major determining factors in
plasmon shift in a simplistic 2-branch model.
[0015] Because existing nanoparticles are associated with
limitations as described above, new nanoparticles with improved
properties are therefore desirable.
SUMMARY OF THE INVENTION
[0016] In one embodiment, a plasmonics-active gold nanostar is
provided resulting from a process comprising: reducing aqueous gold
(Au.sup.3+) to solid gold (Au) in an acidic solution; and mixing a
silver salt compound with a weak reducing agent into the solution
under conditions such that the plasmonics-active gold nanostars are
produced.
[0017] In one embodiment, a method is provided for preparing
plasmonics-active gold nanostars free of polymer, the method
comprising: reducing aqueous gold (Au.sup.3+) to solid gold (Au) in
an acidic solution; and mixing a silver salt compound with a weak
reducing agent into the solution under conditions such that the
plasmonics-active gold nanostars are produced.
[0018] In one embodiment, a method is provided of treating
undesirable cells comprising: contacting an undesirable cell with a
plasmonics-active gold nanostar resulting from a process
comprising: reducing aqueous gold (Au.sup.3+) to solid gold (Au) in
an acidic solution; and mixing a silver salt compound with a weak
reducing agent into the solution under conditions such that the
plasmonics-active gold nanostars are produced; and applying a
single-photon or multi-photon excitation to the undesirable cells
such that the undesirable cells are damaged by one or both of
thermal energy from the single-photon or multi-photon excitation
and thermal energy emitted as a result of excitation of the
nanostar by the single-photon or multi-photon excitation.
[0019] In one embodiment, a method is provided wherein the
plasmonics-active gold nanostar further comprises one or more of a
photosensitizer and a photoactivator, wherein the photosensitizer
and the photoactivator absorb electromagnetic radiation from one or
both of electromagnetic radiation emitted as a result of excitation
of the nanostar and directly from the single-photon or multi-photon
excitation, such that the undesirable cells are damaged by one or a
combination of thermal energy from the single-photon or
multi-photon excitation, thermal energy emitted by the nanostar,
reactive oxygen species (ROS) generated by the photosensiter, and
one or a combination of activation and release of the
photoactivator.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The foregoing summary, as well as the following detailed
description of various embodiments, is better understood when read
in conjunction with the appended figures. For the purposes of
illustration, there is shown in the Figures exemplary embodiments;
however, the presently disclosed subject matter is not limited to
the specific methods and exemplary embodiments disclosed.
[0021] FIGS. 1A-1I are schematic diagrams showing a series of
plasmonics-active nanostars according to one or more embodiments of
the present disclosure. 1A-1H show the plasmonics-active nanostars
and 1I shows the legend.
[0022] FIGS. 2A-2I are schematic diagrams showing a series of
plasmonics-active nanostars with bioreceptor according to one or
more embodiments of the present disclosure. 2A-2H show the
plasmonics-active nanostars and 2I shows the legend.
[0023] FIG. 3 is graph showing the "theranostics window" in tissue
and absorption spectra of biological components.
[0024] FIGS. 4A-4B are schematic diagrams showing a non-invasive
use of a PET- and drug-functionalized nanostar (PET drug) for
photothermal therapy and diagnostics according to one or more
embodiments of the present disclosure.
[0025] FIGS. 5A-5B are schematic diagrams showing a non-invasive
use of a PET- and drug-functionalized nanostar (PET drug) for
photodynamic therapy and diagnostics according to one or more
embodiments of the present disclosure.
[0026] FIG. 6 is a schematic diagram of a synthesis procedure for
forming different nanostars according to one or more embodiments of
the present disclosure. Increasing silver nitrate concentration is
represented as A) 5 .mu.M, B) 10 .mu.M, C) 20 .mu.M, and D) 30
.mu.M.
[0027] FIG. 7 is a series of TEM images of nanostars formed under
different Ag.sup.+ concentrations according to one or more
embodiments of the present disclosure. The top panel shows
increasing Ag.sup.+ concentrations as: (S5) 5 .mu.M, (S10) 10
.mu.M, (S20) 20 .mu.M, and (S30) 30 .mu.M. The bottom panel shows
simulation of |E| in the vicinity of the nanostars in response to a
z-polarized plane wave incident E-field of amplitude 1, propagating
in the y-direction and with a wavelength of 800 nm. The insets
depict the 3-D geometry of the stars. Diagrams are not to
scale.
[0028] FIGS. 8A-8B are absorbance spectra illustrating the
tunability of the nanostars' plasmon peak from 600 nm to 1000 nm
according to one or more embodiments of the present disclosure. (A)
Absorbance spectra (unnormalized) of the star solutions (.about.0.2
nM) in citrate buffer. (inset) Photograph of the corresponding star
solutions. (B) Corresponding FEM-generated absorption spectra
(.+-.1 SD) of nanostars embedded in water. The solved data points
were interpolated with a spline fit. The orientation dependence of
the incident E-field was accounted for by averaging the absorption
spectra of the nanostars as they were incrementally rotated by 30
degrees in the [x=y] plane, such that the orientation of the
branches relative to the z-polarized incident field became
randomized.
[0029] FIG. 9 is a TEM micrograph of hollow gold nanostar according
to one or more embodiments of the present disclosure.
[0030] FIG. 10 is a schematic diagram of a process for synthesizing
a magnetic gold nanostar according to one or more embodiments of
the present disclosure.
[0031] FIG. 11 is a TEM micrograph of the silica coated AuNS
according to one or more embodiments of the present disclosure. The
scale bar is 100 nm.
[0032] FIG. 12 is a fluorescence spectra of AuNS-DTTC@SiO.sub.2-MB
(solid line), MB-spiked AuNS-DTTC@SiO.sub.2 (dotted line), and
AuNS-DTTC@SiO.sub.2 (dashed line) in water according to one or more
embodiments of the present disclosure. Excitation was at 633 nm, 10
sec exposure time.
[0033] FIG. 13 is a SERS spectra (baseline subtracted) of 1 .mu.M
4-MBA in 0.1 nM AgNP, AuNP, and nanostar solutions examined through
a Raman microscope under 785 nm excitation according to one or more
embodiments of the present disclosure. Methanol (10% v/v) was used
as an internal reference. Wavenumber 1013 and 1078 represents Raman
signals from MeOH and 4-MBA, respectively.
[0034] FIG. 14 is a graph showing enhancement factors of AgNP and
nanostar under 785 nm (grey) and 633 nm (white) laser excitation
according to one or more embodiments of the present disclosure.
AuNP was omitted due to no 4-MBA SERS signal available for EF
calculation. Error bar is 1 SD.
[0035] FIG. 15 shows three SERS spectra (baseline subtracted) of
different intracellular regions (top SERS spectrum: cytoplasm;
middle SERS spectrum: nucleus; bottom SERS spectrum: glass) on
BT549 cells incubated 24 hrs with silica-coated nanostars labeled
with a SERS dye (DTTC) according to one or more embodiments of the
present disclosure.
[0036] FIGS. 16A-16B show A) a schematic diagram of NIR-SERRS probe
synthesis and B) a baseline-subtracted SERS spectra from probes
made of 4 different NIR dyes (785 nm excitation, 100 mW, 100 ms)
according to one or more embodiments of the present disclosure.
[0037] FIGS. 17A-17B are SERS spectra of 4-MBA (1 .mu.M) on 0.1 nM
nanospheres A) and nanostars B) examined under 785 nm and 633 nm
excitations according to one or more embodiments of the present
disclosure. 10% v/v methanol was used as an internal reference.
[0038] FIG. 18 is a diagram of a SERS Multispectral detection and
Hyperspectral imaging system for flow cytometry according to one or
more embodiments of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0039] For the purposes of promoting an understanding of the
principles of the present disclosure, reference will now be made to
preferred embodiments and specific language will be used to
describe the same. It will nevertheless be understood that no
limitation of the scope of the disclosure is thereby intended, such
alteration and further modifications of the disclosure as
illustrated herein, being contemplated as would normally occur to
one skilled in the art to which the disclosure relates.
[0040] Articles "a" and "an" are used herein to refer to one or to
more than one (i.e. at least one) of the grammatical object of the
article. By way of example, "a cell" means at least one cell and
can include a number of cells.
[0041] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs.
[0042] As used herein, the term "nanostar" or "NS" means a
nanoparticle which has a single core section with two or more
protrusions emitting from the core section of the nanoparticle.
These protrusions are usually conical or pyramidal in form, but not
always.
[0043] The present disclosure is provided in response to a need to
develop a versatile platform for use in diagnostics, imaging and
disease treatment. In addition, there is also a need for a
combination of both diagnostics and therapy, and the present
disclosure provides a solution referred to herein as
"theranostics". The subject matter disclosed herein describes
fabrication and application of plasmonics-active metallic platforms
such as nanostars for various theranostics applications combining
and including, but not limited to: Surface-enhanced Raman
scattering (SERS) diagnostics and imaging; Enhanced fluorescence
detection using single and two-photon excitation; Enhanced diffuse
scattering diagnostics and imaging; Enhanced optical coherence
tomography (OCT); Enhanced photoacoustics; Enhanced photothermal
therapy; Enhanced photodynamic therapy; Enhanced immunotherapy; and
Enhanced theranostics by combining the above detection modalities
(diagnostics) with therapy modalities.
[0044] Enhancement mechanisms of the electromagnetic field effect
that can increase the absorption of the excitation light used for
optical diagnostics and therapy is the basic mechanism of the NIDT
operating principle. There are two main sources of electromagnetic
enhancement: (1) first, the laser electromagnetic field is enhanced
due to the addition of a field caused by the polarization of the
metal particle; (2) in addition to the enhancement of the
excitation laser field, there is also another enhancement due to
the molecule radiating an amplified emission (luminescence, Raman,
etc.) field, which further polarizes the metal particle, thereby
acting as an antenna to further amplify the Raman/Luminescence
signal.
[0045] Electromagnetic enhancements are divided into two main
classes: a) enhancements that occur only in the presence of a
radiation field, and b) enhancements that occur even without a
radiation field. The first class of enhancements is further divided
into several processes. Plasma resonances on the substrate
surfaces, also called surface plasmons, provide a major
contribution to electromagnetic enhancement. An effective type of
plasmonics-active substrate consists of nanostructured metal
particles, protrusions, or rough surfaces of metallic materials.
Incident light irradiating these surfaces excites conduction
electrons in the metal, and induces excitation of surface plasmons
leading to Raman/Luminescence enhancement. At the plasmon
frequency, the metal nanoparticles (or nanostructured roughness)
become polarized, resulting in large field-induced polarizations
and thus large local fields on the surface. These local fields
increase the Luminescence/Raman emission intensity, which is
proportional to the square of the applied field at the molecule. As
a result, the effective electromagnetic field experienced by the
analyte molecule on theses surfaces is much larger than the actual
applied field. This field decreases as 1/r.sup.3 away from the
surface. Therefore, in the electromagnetic models, the
luminescence/Raman-active analyte molecule is not required to be in
contact with the metallic surface but can be located anywhere
within the range of the enhanced local field, which can polarize
this molecule. The dipole oscillating at the wavelength .lamda. of
Raman or luminescence can, in turn, polarize the metallic
nanostructures and, if .lamda. is in resonance with the localized
surface plasmons, the nanostructures can enhance the observed
emission light (Raman or luminescence). There are two main sources
of electromagnetic enhancement: (1) first, the laser
electromagnetic field is enhanced due to the addition of a field
caused by the polarization of the metal particle; (2) in addition
to the enhancement of the excitation laser field, there is also
another enhancement due to the molecule radiating an amplified
Raman/Luminescence field, which further polarizes the metal
particle, thereby acting as an antenna to further amplify the
Raman/Luminescence signal. Plasmonics-active metal nanoparticles
also exhibit strongly enhanced visible and near-infrared light
absorption, several orders of magnitude more intense compared to
conventional laser phototherapy agents. The use of plasmonic
nanoparticles as highly enhanced photoabsorbing agents has thus
introduced a much more selective and efficient phototherapy
strategy. The tunability of the spectral properties of the metal
nanoparticles and the biotargeting abilities of the plasmonic
nanostructures make the method desireable.
[0046] The methods and compositions of the present disclosure are
based on several important mechanisms: Increased absorption of the
excitation light by the plasmonic metal nanoplatforms (i.e.,
nanostars) resulting in enhanced absorption of the nanoplatforms;
Increased absorption of the excitation light by the plasmonic metal
nanoplatforms (i.e., nanostars), yielding more light for excitation
of optical labels (Raman, fluorescence, etc); Increased absorption
of the excitation light by the plasmonic metal nanoplatforms (i.e.,
nanostars), resulting in increased photothermal heating of the
plasmonic metal nanoplatforms (i.e., nanostars); Increased
absorption of the excitation light by the dye (Raman, fluorescent,
phosphorescent labels, etc) adsorbed on or near the plasmonic metal
nanoplatforms (i.e., nanostars); Increased light absorption of the
dye label molecules adsorbed on or near the metal nanoplatforms
(i.e., nanostars); Amplified emission from the dye label and/or
photodynamic molecules adsorbed on or near the metal nanoparticles;
and Combination of enhanced detection and enhanced therapy via the
above processes.
[0047] One of several phenomena that can enhance the efficiency of
light emitted (Raman or luminescence) from molecules adsorbed or
near a metal nanostructures Raman scatter is the SERS effect
discovered in the 1970s [Fleischmann; Hendra, P. J.; McQuillan A.
J. Chem. Phys. Lett. 1974, 26 (2), 163-166; Jeanmaire, D. L.;
Vanduyne, R. P. J. Electroanal. Chem. 1977, 84 (1), 1-20; Albrecht,
M. G.; Creighton, J. A. J. Am. Chem. Soc. 1977, 99 (15),
5215-5217]. In 1984, the Vo-Dinh's laboratory first reported the
general applicability of SERS as an analytical technique,_and the
possibility of SERS measurement for a variety of chemicals
including several homocyclic and heterocyclic polyaromatic
compounds [T. Vo-Dinh, M. Y. K. Hiromoto, G. M. Begun and R. L.
Moody, "Surface-enhanced Raman spectroscopy for trace organic
analysis," Anal. Chem., vol. 56, 1667, 1984]. Extensive research
has been devoted to understanding and modeling the Raman
enhancement in SERS since the mid 1980's. The intensity of the
normally weak Raman scattering process is increased by factors as
large as 10.sup.13 or 10.sup.15 for compounds adsorbed onto "hot
spots" on a plasmonics-active substrate, allowing for
single-molecule detection. As a result of the electromagnetic field
enhancements produced near nanostructured metal surfaces,
nanoparticles have found increased use as fluorescence and Raman
nanoprobes.
[0048] The theoretical models indicate that it is possible to tune
the size of the nanoparticles and the nanoshells to the excitation
wavelength. Experimental evidence suggests that the origin of the
10.sup.6- to 10.sup.15-fold Raman enhancement primarily arises from
two mechanisms: a) an electromagnetic "lightning rod" effect
occurring near metal surface structures associated with large local
fields caused by electromagnetic resonances, often referred to as
"surface plasmons"; and b) a chemical effect associated with direct
energy transfer between the molecule and the metal surface.
[0049] According to classical electromagnetic theory,
electromagnetic fields can be locally amplified when light is
incident on metal nanostructures. These field enhancements can be
quite large (typically 10.sup.6- to 10.sup.7-fold, but up to
10.sup.15-fold enhancement at "hot spots"). When a nanostructured
metallic surface is irradiated by an electromagnetic field (e.g., a
laser beam), electrons within the conduction band begin to
oscillate at a frequency equal to that of the incident light. These
oscillating electrons, called "surface plasmons," produce a
secondary electric field, which adds to the incident field. If
these oscillating electrons are spatially confined, as is the case
for isolated metallic nano spheres or roughened metallic surfaces
(nanostructures), there is a characteristic frequency (the plasmon
frequency) at which there is a resonant response of the collective
oscillations to the incident field. This condition yields intense
localized field enhancements that can interact with molecules on or
near the metal surface. In an effect analogous to a "lightning
rod," secondary fields are typically most concentrated at points of
high curvature on the roughened metal surface.
[0050] FIGS. 1A-1H are schematic diagrams showing various
plasmonics-active nanostars according to one or more embodiments of
the present disclosure. FIG. 1I shows the legend for FIGS. 1A-1H.
FIG. 1A shows a plasmonics-active nanostar. FIG. 1B shows the
nanostar labeled with optical dye and/or a drug molecule. FIG. 1C
shows the nanostar having a layer embedded with a label and/or a
drug. FIG. 1D shows the nanostar with a layer embedded with a label
and/or drug and a protective overlayer. FIG. 1E shows the nanostar
with a paramagnetic spherical nucleus. FIG. 1F shows the nanostar
with an elongated paramagnetic nucleus. FIG. 1G shows the nanostar
having a void-space. FIG. 1H shows the nanostar having an empty or
dielectric core.
[0051] In another aspect of the present disclosure, the nanostars
can include bioreceptors that can be used for specificity for
targeting disease cells. The bioreceptors can be responsible for
binding the nanostar to the biotarget of interest for therapy.
These bioreceptors can take many forms and the different
bioreceptors that can be used are as numerous as the different
analytes that have been monitored using biosensors. Bioreceptors
can generally be classified into five different major categories.
These categories include: 1) antibody/antigen, 2) enzymes, 3)
nucleic acids/DNA, 4) cellular structures/cells and 5) biomimetic
(aptamers, peptides, etc).
[0052] FIGS. 2A-2H are schematic diagrams showing various
plasmonics-active nanostars with bioreceptor according to one or
more embodiments of the present disclosure. FIG. 2I shows the
legend for FIGS. 2A-2H. The nanostars shown in FIGS. 2A-2H are
similar to those shown in FIGS. 6A-H but also have a bioreceptor
for targeting to a specific cell or a tumor. FIG. 2A shows a
plasmonics-active nanostar with bioreceptor. FIG. 2B shows the
nanostar labeled with optical dye and/or a drug molecule with
bioreceptor. FIG. 2C shows the nanostar having a layer embedded
with a label and/or a drug (e.g., psoralen) with bioreceptor. FIG.
2D shows the nanostar with a layer embedded with a label and/or
drug (e.g., psoralen) and a protective overlayer with bioreceptor.
FIG. 2E shows the nanostar with a paramagnetic spherical nucleus
with bioreceptor. FIG. 2F shows the nanostar with an elongated
paramagnetic nucleus with bioreceptor. FIG. 2G shows the nanostar
having a void-space with bioreceptor. FIG. 2H shows the nanostar
having an empty or dielectric core with bioreceptor.
[0053] To specifically target diseases cells, specific genes or
protein markers, the nanostars of the present disclosure can be
bound to a bioreceptor (e.g., antibody, DNA, proteins, cell-surface
receptors, aptamers, etc.) as described above. A general
description of certain of the bioreceptors is provided below.
[0054] DNA Probes.
[0055] The operation of gene probes is based on the hybridization
process. Hybridization involves the joining of a single strand of
nucleic acid with a complementary probe sequence. Hybridization of
a nucleic acid probe to DNA biotargets (e.g., gene sequences of a
mutation, etc) offers a very high degree of accuracy for
identifying DNA sequences complementary to that of the probe.
Biologically active DNA probes can be directly or indirectly
immobilized onto a drug system, such as the EEC system (e.g., gold
nanoparticle, a semiconductor, quantum dot, a glass/quartz
nanoparticles, etc.) surface to ensure optimal contact and maximum
binding. When immobilized onto nanoparticles including nanostars,
the gene probes are stabilized and, therefore, can be reused
repetitively. Several methods can be used to bind DNA to different
supports. The method commonly used for binding DNA to glass
involves silanization of the glass surface followed by activation
with carbodiimide or glutaraldehyde. The silanization method can be
used for binding to glass surfaces using
3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS) to covalently link DNA via amino
linkers incorporated either at the 3' or 5' end of the molecule
during DNA synthesis.
[0056] Antibody Probes.
[0057] Antibodies are biological molecules that exhibit very
specific binding capabilities for specific structures and that can
be used as bioreceptors. This unique property of antibodies is the
key to their usefulness in immunosensors where only the specific
analyte of interest, the antigen, fits into the antibody binding
site.
[0058] Enzyme Probes.
[0059] Enzymes are often chosen as bioreceptors based on their
specific binding capabilities as well as their catalytic activity.
In biocatalytic recognition mechanisms, the detection is amplified
by a reaction catalyzed by macromolecules called biocatalysts. The
catalytic activity provided by enzymes allows for much lower limits
of detection than would be obtained with common binding techniques.
Enzyme-coupled receptors can also be used to modify recognition
mechanisms.
[0060] Other Approaches.
[0061] Methods for conjugation of nanostars with receptor-binding
molecules can be used that can selectively increase the adherence
or uptake of nanostars for targeting cells. In addition to
bioreceptor molecules such as antibodies, antibody fragments, and
DNA/RNA aptamers, peptides can also be used since they offer
several advantages as bioreceptors for nanostars (low cost, high
activity per unit, excellent stability, long-term storage and easy
handling). An enzyme-mediated process can also be used for
targeting. Overexpression of certain enzymes at the site of disease
can be used for the development of enzyme-responsive nanoplatforms
diagnosis. For in vivo models, it is also important keep the
nanoparticles out of the blood circulation to prevent clearance.
The concept of using iron oxide-gold core-shell particles, can
provide a unique solution. The gold shell will allow for the same
functionalization methods to be used from the ex vivo work, while
the iron oxide core will be superparamagnetic. A magnet can be used
to collect and keep the particles at one location in the body, at
which the analysis can be performed. The iron oxide core can
provide for multimodality diagnostics (SERS, luminescence, MRI) and
co-registration.
[0062] Bioreceptors (and other biomolecules) as well as drug
molecules can be immobilized to a solid support such as a metal
nanostar using a wide variety of methods published in the
literature. Binding can be performed through covalent bonds by
taking advantage of reactive groups such as amine (--NH.sub.2) or
sulfide (--SH) that are naturally present or can be incorporated
into the bioreceptor/biomolecule structure. For example, amines can
react with carboxylic acid or ester moieties in high yield to form
stable amide bonds. Thiols can participate in maleimide coupling,
yielding stable dialkylsulfides.
[0063] A solid support of interest is gold (or silver)
nanoparticles. The majority of immobilization schemes involving
Au(Ag) surfaces utilize a prior derivatization of the surface with
alkylthiols, forming stable linkages. Alkylthiols readily form
self-assembled monolayers (SAM) onto silver surfaces in micromolar
concentrations. The terminus of the alkylthiol chain can be used to
bind biomolecules, or can be easily modified to do so. The length
of the alkylthiol chain has been found to be an important
parameter, keeping the biomolecules away from the surface.
Furthermore, to avoid direct, non-specific DNA adsorption onto the
surface, alkylthiols have been used to block further access to the
surface, allowing only covalent immobilization through the linker
[Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70,
4670-7; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119,
8916-20].
[0064] Silver surfaces have been found to exhibit controlled
self-assembly kinetics when exposed to dilute ethanolic solutions
of alkylthiols. The tilt angle formed between the surface and the
hydrocarbon tail ranges from 0 to 15.degree.. There is also a
larger thiol packing density on silver, when compared to gold
[Burges, J. D.; Hawkridge, F. M. Langmuir 1997, 13, 3781-6]. After
SAM formation on gold/silver nanoparticles, alkylthiols can be
covalently coupled to biomolecules. The majority of synthetic
techniques for the covalent immobilization of biomolecules utilize
free amine groups of a polypeptide (enzymes, antibodies, antigens,
etc) or of amino-labeled DNA strands, to react with a carboxylic
acid moiety forming amide bonds. As a general rule, a more active
intermediate (labile ester) is first formed with the carboxylic
acid moiety and in a later stage reacted with the free amine,
increasing the coupling yield. Successful coupling procedures
include:
[0065] Binding Procedure Using N-Hydroxysuccinimide (NHS) and its
Derivatives.
[0066] The coupling approach involves the esterification under mild
conditions of a carboxylic acid with a labile group, an
N-hydroxysuccinimide (NHS) derivative, and further reaction with
free amine groups in a polypeptide (enzymes, antibodies, antigens,
etc) or amine-labeled DNA, producing a stable amide [Boncheva, M.;
Scheibler, L.; Lincoln, P.; Vogel, H.; Akerman, B. Langmuir 1999,
15, 4317-20]. NHS reacts almost exclusively with primary amine
groups. Covalent immobilization can be achieved in as little as 30
minutes. Since H.sub.2O competes with --NH.sub.2 in reactions
involving these very labile esters, it is important to consider the
hydrolysis kinetics of the available esters used in this type of
coupling. The derivative of NHS used in FIG. 1,
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate,
increases the coupling yield by utilizing a leaving group that is
converted to urea during the carboxylic acid activation, hence
favorably increasing the negative enthalpy of the reaction.
[0067] Binding Procedure Using Maleimide.
[0068] Maleimide can be used to immobilize biomolecules through
available thiol moieties. Coupling schemes with maleimide have been
proven useful for the site-specific immobilization of antibodies,
Fab fragments, peptides, and SH-modified DNA strands. Sample
preparation for the maleimide coupling of a protein involves the
simple reduction of disulfide bonds between two cysteine residues
with a mild reducing agent, such as dithiothreitol,
2-mercaptoethanol or tris(2-carboxyethyl)phosphine hydrochloride.
However, disulfide reduction will usually lead to the protein
losing its natural conformation, and might impair enzymatic
activity or antibody recognition. The modification of primary amine
groups with 2-iminothiolane hydrochloride (Traut's reagent) to
introduce sulfydryl thiol groups is an alternative for biomolecules
lacking them. Free sulfhydryl thiols are immobilized to the
maleimide surface by an addition reaction to unsaturated
carbon-carbon bonds [Jordan, C. E., et al., 1997].
[0069] Binding Procedure Using Carbodiimide.
[0070] Surfaces modified with mercaptoalkyldiols can be activated
with 1,1'-carbonyldiimidazole (CDI) to form a carbonylimidazole
intermediate. A biomolecule with an available amine group displaces
the imidazole to form a carbamate linkage to the alkylthiol
tethered to the surface [Potyrailo, R. A., et al., 1998].
[0071] Non-Invasive Photon Excitation Modalities of Nanostars in
the NIR "Theranostic Window". Photon Excitation in the Therapeutic
Window of Tissue. There are several methods of using light having
wavelengths within the so-called "diagnostic window" or
"therapeutic window" (700-1300 nm) to excite and photoactivate
compounds in tissue in a non-invasive manner. Since nanostars can
be used as platforms for both diagnostic and therapy, this spectral
region is referred to herein as "theranostic window". The ability
of light to penetrate tissues depends on absorption. Within the
spectral range known as the therapeutic window (or diagnostic
window), most tissues are sufficiently weak absorbers to permit
significant penetration of light. This window extends from 600 to
1300 nm, from the orange/red region of the visible spectrum into
the NIR. At the short-wavelength end, the window is bound by the
absorption of hemoglobin, in both its oxygenated and deoxygenated
forms. The absorption of oxygenated hemoglobin increases
approximately two orders of magnitude as the wavelength shortens in
the region around 600 nm. At shorter wavelengths many more
absorbing biomolecules become important, including DNA and the
amino acids tryptophan and tyrosine. At the infrared (IR) end of
the window, penetration is limited by the absorption properties of
water. Within the therapeutic window, scattering is dominant over
absorption, and so the propagating light becomes diffuse, although
not necessarily entering into the diffusion limit. FIG. 3 shows a
diagram of the therapeutic window of tissue. The following section
discusses the use of one-photon and multi-photon techniques for
therapy.
[0072] Two methods can be used for therapy, single-photon or
multi-photon excitation. FIGS. 4A-4B are schematic diagrams showing
the non-invasive use of a functionalized nanostar for photothermal
therapy according to one or more embodiments of the present
disclosure. In one example, nanostars of the present disclosure are
functionalized with a positron emission tomography (PET) label and
with a drug molecule. The plasmonics-enhanced theranostics (PET)
drug molecules are given to a patient by oral ingestion or by
intravenous injection. The PET drugs travel through the blood
stream inside the body towards the targeted tumor (either via
passive or active targeting strategies). If the disease is
systematic in nature a photon radiation at a suitable wavelength
such as, for example, radio frequency (RF), microwave (MW), infra
red (IR), NIR, VIS, UV, and X ray can be used to irradiate the skin
of the patient, the light being selected to penetrate deep inside
the patient's tissue (e.g., NIR). For solid tumors, the radiation
light source can be directed at the tumor. Subsequently, a
treatment procedure can be initiated using delivery of energy into
the tumor site. One or several light sources may be used as
described herein. One example of therapy consists of sending NIR
radiation using an NIR laser through focusing optics. The heat can
be used to kill diseased cells or tissues. Alternatively, the heat
can be used to release psoralen (or another drug of choice).
[0073] FIGS. 5A-5B are schematic diagrams showing the non-invasive
use of a functionalized nanostar for photodynamic therapy (PDT)
according to one or more embodiments of the present disclosure. In
one example, nanostars of the present disclosure are functionalized
with a PET label and with a drug molecule for photodynamic therapy
such as, for example, a photosensitizer or a photoactivator
molecule. The plasmonics-enhanced theranostics (PET) drug molecules
are given to a patient by oral ingestion or by intravenous
injection. The PET drugs travel through the blood stream inside the
body towards the targeted tumor (either via passive or active
targeting strategies). If the disease is systematic in nature a
photon radiation at a suitable wavelength such as, for example,
radio frequency (RF), microwave (MW), infra red (IR), NIR, VIS, UV,
and X ray can be used to irradiate the skin of the patient, the
light being selected to penetrate deep inside the patient's tissue
(e.g., NIR). For solid tumors, the radiation light source can be
directed at the tumor. Subsequently, a treatment procedure can be
initiated using delivery of energy into the tumor site. One or
several light sources may be used as described herein. One example
of therapy consists of sending NIR radiation using an NIR laser
through focusing optics. The photodynamic molecule can be used to
kill diseased cells or tissues.
[0074] Table 1 shows some examples of the plasmonics-active
nanostar methods of the present disclosure that combine diagnostics
and therapy (Theranostics) using optical and non-optical
techniques.
TABLE-US-00001 TABLE 1 Examples of Theranostics Methods Treatment
Methods Other Phototherapy Photothermal optical treatments (e.g.,
Psoralen) therapy (e.g., ROS) Detection Methods Fluorescence (1-p,
x x x 2-p, multi-p) Phosphorescence x x x Raman x x x Diffuse
Scattering x x x Absorption x x x Optical Coherence x x x Methods
Photoacoustics x x x X-ray x x x MRI x x x PET x x x
[0075] Focused beam or other radiation including but not limited to
such as, for example, X ray, MW, and RF can also be used and will
depend upon the treatment methods used. For X-ray excitation, the
core of the nanostars can consist of materials that exhibit X-ray
excited luminescence (XEOL). There is a wide variety of materials
that exhibit XEOL including but not limited to such as, for
example, organic, inorganic solids, crystals, lanthanides,
polymers.
[0076] In one embodiment of the present disclosure,
plasmonics-active gold nanostars are provided that result from a
process that includes reducing aqueous gold (Au.sup.3+) to solid
gold (Au) in an acidic solution; and mixing a silver salt compound
with a weak reducing agent into the solution under conditions such
that the plasmonics-active gold nanostars are produced.
[0077] In one embodiment, plasmonics-active gold nanostars are
provided that result from a process that consists of reducing
aqueous gold (Au.sup.3+) to solid gold (Au) in an acidic solution;
and mixing a silver salt compound with a weak reducing agent into
the solution under conditions such that the plasmonics-active gold
nanostars are produced.
[0078] In one embodiment, a method is provided for preparing
plasmonics-active gold nanostars free of polymer that includes
reducing aqueous gold (Au.sup.3+) to solid gold (Au) in an acidic
solution; and mixing a silver salt compound with a weak reducing
agent into the solution under conditions such that the
plasmonics-active gold nanostars are produced.
[0079] In one embodiment, a method is provided for preparing
plasmonics-active gold nanostars free of polymer that consists of
reducing aqueous gold (Au.sup.3+) to solid gold (Au) in an acidic
solution; and mixing a silver salt compound with a weak reducing
agent into the solution under conditions such that the
plasmonics-active gold nanostars are produced.
[0080] The Au.sup.3+ can comprise tetrachloroauric acid
(HAuCl.sub.4). The Au.sup.3+ can consist essentially of
tetrachloroauric acid (HAuCl.sub.4). The Au.sup.3+ can be reduced
to solid Au onto citrate-stabilized gold seeds.
[0081] The citrate-stabilized gold seeds used in the method can
include or can consist essentially of hollow gold nanoshells and
the plasmonics-active gold nanostars that are produced in the
method can be hollow. The citrate-stabilized gold seeds used in the
method can include or can consist essentially of superparamagnetic
particles coated with a layer of gold, and the plasmonics-active
gold nanostars that are produced in the method can be
superparamagnetic. The superparamagnetic particles used in the
method can include or can consist essentially of iron oxide
(IO).
[0082] The weak reducing agent can consist essentially of ascorbic
acid. The silver salt compound can consists essentially of silver
nitrate (AgNO.sub.3). The concentration of silver cation of the
silver compound can range from about 5 .mu.M to about 30 .mu.M and
the plasmon peak of the nanostar can range from about 600 nm to
about 1000 nm. The ratio of the ascorbic acid to the HAuCl.sub.4
can range from about 1.5 to about 2. The nanostars can have a size
of less than about 100 nm.
[0083] In one embodiment, the plasmonics-active gold nanostars can
have one or more of an optical label, a photosensitizer, and a
photoactivator. The optical label can include, for example, but is
not limited to one or more of a fluorescence label, fluorescein,
fluorescein isothiocyanate (FITC), thionine dyes, rhodamine,
crystal violet, Raman label, 3,3'-diethylthiatricarbocyanine iodide
(DTTC), absorbance label, positively-charged hydrophobic NIR dyes,
IR-780, IR-792, IR-797, IR-813, methylene blue hydrate (MB),
4-mercaptobenzoic acid (4-MBA), 5,5'-dithiobis-2-nitrobenzoic acid
(DTNB), and 4-aminothiophenol (4ATP).
[0084] In one embodiment, the nanostar can include one or both of a
layer surrounding the nanostar having within the layer the one or
more of the optical label, the photosensitizer, and the
photoactivator, and a protective overlayer surrounding the
layer.
[0085] In one embodiment, a method is provided for treating
undesirable cells that includes contacting undesirable cells with
the plasmonics-active gold nanostars described above, and applying
a single-photon or multi-photon excitation to the undesirable cells
such that the undesirable cells are damaged by one or both of
thermal energy from the single-photon or multi-photon excitation
and thermal energy emitted as a result of excitation of the
nanostar by the single-photon or multi-photon excitation.
[0086] In one embodiment, in the method the plasmonics-active gold
nanostar can further include one or more of a photosensitizer and a
photoactivator, wherein the photosensitizer and the photoactivator
absorb electromagnetic radiation from one or both of
electromagnetic radiation emitted as a result of excitation of the
nanostar and directly from the single-photon or multi-photon
excitation, such that the undesirable cells are damaged by one or a
combination of thermal energy from the single-photon or
multi-photon excitation, thermal energy emitted by the nanostar,
reactive oxygen species (ROS) generated by the photosensiter, and
one or a combination of activation and release of the
photoactivator. The photosensitizer can include, for example, but
is not limited to a porphyrin, a Protoporphyrin IX, or a methylene
blue. The photoactivator can include, for example, but is not
limited to a psoralen or a psoralen variant.
[0087] In one embodiment of the method, the plasmonics-active
nanostar can further include an optical label that absorbs
electromagnetic radiation from one or both of electromagnetic
radiation emitted as a result of excitation of the nanostar and
directly from the single photon or multi-photon excitation such
that the optical label emits detectable electromagnetic radiation.
The optical label can include, for example, but is not limited to
one or more of a fluorescence label, fluorescein, fluorescein
isothiocyanate (FITC), thionine dyes, rhodamine, crystal violet, a
Raman label, 3,3'-diethylthiatricarbocyanine iodide (DTTC), an
absorbance label, a positively-charged hydrophobic near infrared
(NIR) dye, IR-780, IR-792, IR-797, IR-813, methylene blue hydrate
(MB), 4-mercaptobenzoic acid (4-MBA), 5,5'-dithiobis-2-nitrobenzoic
acid (DTNB), and 4-aminothiophenol (4ATP).
[0088] In one embodiment, the method can further include detecting
the electromagnetic radiation emitted by the optical label by one
or more of fluorescence detection, surface enhanced Raman
scattering (SERS) detection, surface-enhanced resonance Raman
scattering (SERRS), and absorbance detection.
[0089] In one embodiment of the method, the nanostar can include
one or both of a layer surrounding the nanostar having within the
layer the one or more of the optical label, the photosensitizer,
and the photoactivator, and a protective overlayer surrounding the
layer, such that the optical label, the photosensitizer, and the
photoactivator can be released or activated via one or more of
passive diffusion release, photochemically triggered release,
thermal triggered release, pH triggered release, photochemical
activation, and thermal activation.
[0090] The undesireable cells can be cells present in a tissue or
the undesireable cells can be present in a subject such as, for
example, a patient or an animal. In one embodiment of the method,
the single-photon or multi-photon excitation can be applied to the
tissue or to the subject at an irradiance of about 0.2-0.4
W/cm.sup.2 at about 700-900 nm. The undesireable cells can be
cancer cells.
EXAMPLES
[0091] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject
matter.
Example 1
Polymer-Free Synthesis Method for Preparation of High-Yield
Monodisperse Gold Nanostars
[0092] Recently, star-shaped AuNPs ("nanostars") have attracted
interest because their plasmon can be tuned to the NIR region, and
the structure contains multiple sharp tips that can greatly enhance
incident electromagnetic fields. Studies have shown that
NIR-absorbing nanorods, nanocages or nanoshells can be used as
contrast agents in optical imaging techniques such as optical
coherent tomography, two-photon luminescence (TPL) microscopy, and
photoacoustic imaging. Their large absorption cross-sections can
also effectively convert photon energy to heat during photothermal
therapy. Nanostars, which absorb in the NIR, are hypothesized to
behave similarly. Nanostar-related bioapplication remain scarce in
spite of their potential, mostly due to the difficulty of surface
functionalization.
[0093] In 2003, Chen et al. [[Chen S, Wang Z L, Ballato, Foulger S
H, Carroll D L. J Am Chem. Soc. 2003 Dee 31; 125(52):16186-7]]
first reported the synthesis of multipod gold nanoparticles from
silver plates in the presence of cetyltrimethylammonium bromide
(CTAB) and NaOH. Later, several seedless or seed-mediated synthesis
methods were employed using majorly poly(N-vinylpyrolidone) (PVP)
or CTAB as surfactant. Further use of nanostars has been limited by
(1) the potential toxicity of CTAB, (2) the difficulty of replacing
PVP or CTAB during biofunctionalization, and (3) induction of
aggregation following multiple washes. A polymer-free synthesis can
potentially circumvent these issues. In the meantime, previous
experimental studies have shown a red-shifting of the plasmon peak
from nanostars with longer or sharper branches. Several numerical
studies of their plasmonic properties have recently been reported.
Hao et al.'s [[Hao F, Nehl C L, Hafner Nordlander P. Nano Lett.
2007 March; 7(3):729-32]] 3-D modeling of a single nanostar,
consisting of 5 unique branches, with finite difference time domain
(FDTD) method showed that nanostars plasmon results from the
hybridization of plasmon resonance of each branch; the plasmon peak
relative intensity depends on the polarization angle. Senthil et
al. [Senthil Kumar P, Pastoriza-Santos I, Rodriguez-Gonzalez B,
Garcia de Abajo F J, Liz-Marzan LM. Nanotechnology,
2008:19(1):015606-12] also stated that the tip angle and radius,
but not the number of branches, are the major determining factors
in plasmon shift in a simplistic 2-branch model. However, to
properly determine the effect of nanostar geometry on the plasmon
band, a polarization-averaged multi-branched 3-D nanostar model is
necessary.
[0094] Below is provided a new seed-mediated polymer-free synthesis
method that produces high-yield monodisperse gold nanostars with a
mean tip-to-tip diameter from 50-70 nm. These nanostars have
plasmon bands tunable in the NIR, and use of citrate for
stabilization simplifies surface modification for further
applications. Their optical properties and plasmonic tunability
have been experimentally examined and compared to
polarization-averaged 3-D finite element method (FEM) simulation
results. Finally, use of nanostars as a strong multiphoton contrast
agent during in vitro cellular imaging was investigated.
[0095] Anisotropic Growth of Au Branches on Nanostars.
[0096] Nanostars were produced by reducing tetrachloroauric acid
onto 12-nm citrate-stabilized gold seeds in an acidic environment
using a weak reducing agent, ascorbic acid (AA) (FIG. 6). The
synthesis can be rapid, reproducible and does not require a polymer
as surfactant. Unlike previous methods which take more than hours
of synthesis, the growth of nanostars using this method can be
completed in less than half a minute and the particles can be
stable at 4.degree. C. for at least a week after centrifugal
washing. The method is the simplest and quickest nanostars
synthesis to date. The polymer-free synthesis method effectively
simplifies surface functionalization of the nanostars.
[0097] In order to obtain nanostars of different geometry while
keeping the particle size in a similar range, multiple factors were
investigated, including pH, vortexing speed, and concentration of
AgNO.sub.3, AA, HAuCl.sub.4 and seed. In general, nanostars formed
the most red-shift plasmon under lower pH, higher vortexing speed
and AA/HAuCl.sub.4 ratio 1.5-2. Concentration of HAuCl.sub.4 and
seeds was selected so the resulting nanostars sizes were around 60
nm. Importantly, silver ions play a major role in controlling the
formation of the star geometry. Without adding Ag.sup.+ during
synthesis, the resulting particles were polydisperse in both size
and shape. The addition of a small amount of Ag.sup.+ led to
high-yield monodisperse star-shape particles. The overall particles
diameters synthesized under different Ag.sup.+ concentrations were
within 50-70 nm. Under higher Ag.sup.+ concentration, sharper and
more numerous branches were formed, observable in the TEMs of FIG.
7. Without wishing to be bound to any particular theory of
mechanism, the major role of Ag.sup.+ is not to form Ag branches
but to assist the anisotropic growth of Au branches on
multi-twinned citrate seeds, but not single crystalline CTAB seeds,
through several possible mechanisms that have been reported for the
formation of nanorods, bipyramids and nanostars.
[0098] Plasmon Tunability.
[0099] Plasmon tunability was achieved by adjusting the Ag.sup.+
concentration in the study. Specifically, higher concentrations of
Ag.sup.+ progressively red-shifted the plasmon band of the
nanostars. From TEM images in FIG. 7, higher Ag.sup.+
concentrations lead to the formation of longer, sharper, and more
numerous branches. Looking at FIG. 7, S5 consists of a few
protrusions, while S30 comprises multiple long, sharp branches that
appear to branch even further. The overall size is less than 100
nm, which is smaller than previously reported nanostars. FIG. 8
illustrates the nanostars' plasmon peak being tunable from 600 nm
to 1000 nm by adjusting the Ag.sup.+ concentration. This was
accompanied by a visible change in the solution color from dark
blue to dark grey as the plasmon red-shifts and broadens. Both the
plasmon peak position and spectral width (as defined by the full
width at half maximum (FWHM) of the plasmon peak) followed a linear
trend with increasing Ag.sup.+ concentration. A plateau was reached
around a Ag.sup.+ concentration of 30 .mu.M in this study.
Nanostars can therefore be synthesized in a controlled fashion and
applied in NIR applications.
[0100] Numerical Simulations.
[0101] A simulation that models and compares the optical properties
of different nanostars was performed. Instead of modeling the
plasmon of a single polarization, this analysis featured
polarization-averaging over space as the nanostars were discretely
rotated at 6 angles, a feature that has not been addressed so far.
The 3-D nanostar simulations were performed using the finite
element method (FEM), which yields solutions to the local field
around 3-D metallic nanostructures that are in excellent agreement
with theory. For each of the 4 nanostars, the local E-field is most
greatly enhanced at the tips of those branches that are at least
partially aligned parallel to the incident polarization, most
clearly observable for the top-most branch that is aligned parallel
to the polarization (data not shown). Notably, the E-field at the
surface of all branches is enhanced to a value of at least between
1 and 4, suggesting that these surfaces also contribute to the
total E-field enhancement around the nanostar.
[0102] It was demonstrated here that the experimental absorption
peak shifts that were observed can be modeled by designing a 3-D
nanostar geometry according to the parameters including core
diameter, branch base width, branch length and tip radius. The
modeled absorption peaks of the various nanostars align well with
the experimentally-measured spectra, and reproduce experimentally
observed peaks for each nanostars solution sample (data not shown).
A weak absorption around 520 nm was attributed to the plasmon
resonance of the nanostar's core, and a dominant plasmon band at
longer wavelengths was observed due to the resonance supported by
the nanostar branches. As the number of branches increases from 4
to 10, the peak absorption cross-section increase from S5 to S30
(see FIG. 7 top), respectively. Moreover, the deviation from the
average intensity from average decreases with increasing branch
number (see FIG. 7 top).
[0103] Materials.
[0104] Gold(III) chloride trihydrate (HAuCl.sub.4), sodium citrate
tribasic dihydrate, L(+)ascorbic acid (AA), 4-mercaptobenzoic acid
(pMBA), silver nitrate (AgNO.sub.3), hydrochloric acid (HCl),
methanol (MeOH),
0-(3-Carboxypropyl)-O'-[2-(3-mercaptopropionylamino)-ethyl]-polye-
thylene glycol (Mw 5000; PEG5000), Triticum vulgaris lectin
(wheat-germ agglutinin; WGA) and rhodamine B were purchased from
Sigma-Aldrich (St. Louis, Mo.). DAP1, FM 1-43, RPMI 1640, insulin,
fetal bovine serum were purchased from Invitrogen (Carlsbad,
Calif.). 16% paraformaldehyde was purchased from Alfa Aesar (Ward
Hill, Mass.). (Ortho-pyridyl)disulfide PEG2000-Succinimidyl Ester)
(Mw 2000; OPSS-PEG2000-NHS) was purchased from Creative PEGWorks
(Winston Salem, N.C.). All chemicals were used as received.
Millipore Synergy ultrapure water (resistivity=18.2 M.OMEGA. cm)
was used in all aqueous solutions. All glassware and stir bars were
cleaned with aqua regia solution and oven-dried before use.
[0105] Synthesis of Au Seeds.
[0106] A modified Turkevich method was used to prepare a seed
solution. Briefly, 10 ml of a 38.8 mM citrate solution was added
into 100 ml of a boiling 1 mM HAuCl.sub.4 solution under vigorous
stirring while keeping the volume remained roughly unchanged. After
boiling for about 30 min, the solution was cooled, filtered by a
0.22 micron nitrocellulose membrane, and kept at 4.degree. C. for
long-term storage.
[0107] Synthesis of Au Nanostars.
[0108] Nanostars were prepared by a seed-mediated growth method.
Briefly, 100 .mu.l of the above seed solution (12.+-.0.7 nm;
A.sub.520: 2.81) was added to 10 ml of 0.25 mM HAuCl.sub.4 solution
(with 10 .mu.l of IN HCl) in a 20 ml glass vial at room temperature
under vigorous stirring. Quickly, 100 .mu.l AgNO.sub.3 at different
concentrations (5 to 30 .mu.M) and 50 .mu.l of AA (100 mM) were
added simultaneously. The solution was stirred for 30 sec as its
color rapidly turned from light red to blue or greenish-black.
Immediately afterwards, two centrifugal washes at 3000-5000 rcf
were performed in 15 ml tube to halt the nucleation. The solution
was redispersed in 2 mM pH7 citrate buffer, filtered by a 0.22
.mu.m nitrocellulose membrane, and then kept at 4.degree. C. for
long-term storage.
[0109] Instrumentation.
[0110] The structural features of the nanostars were characterized
using transmission electronic microscopy (TEM; Fei Tecnai G.sup.2
Twin, 200 kV) and analyzed using ImageJ software (National
Institute of Health). The particle hydrodynamic size distribution
and concentration were determined by nanoparticle tracking analysis
(NTA 2.1; build 0342) using NanoSight NS500 (Nanosight Ltd. UK).
Absorbance spectra were obtained using a dual-beam
spectrophotometer (Shimadzu UV-3600; Shimadzu corporation, Japan)
or a microplate reader (BMG LABTECH FLUOStar Omega; BMG LABTECH
GmBH, Germany). More than five samples were analyzed for each
synthesis condition. Morphological features were assessed from more
than 50 particles on different TEM images. Data values were
represented by mean.+-.1 standard deviation.
[0111] Modeling.
[0112] The 3-D nanostar simulations were performed using the finite
element (FEM) based Comsol Multiphysics v3.4 software package and
the RF module (Comsol, Inc. Burlington Mass., USA). A unique, 3-D
nanostar model was designed for each of the samples S5, S10, S20
and S30 using the dimensions obtained from their corresponding TEM
images (data not shown). For a particular star model, the branches
protrude normal to the core surface, but were randomly positioned
on the core such as to maximize the inter-branch distance.
[0113] The dielectric function of gold was modeled using the
Lorentz-Drude model for gold from Johnston and Christy's and the
surrounding medium was modeled as water with a refractive index
n=1.33. The computational domain was bounded by a spherical
perfectly matched layer (PML) to prevent any reflections back onto
the nanoparticle. The nanostars were excited with a z-polarized
incident plane wave of E-field amplitude 1, propagating along the
y-axis and of wavelength ranging 300 nm to 1200 nm. The
nanoparticles were meshed such that the largest mesh size on the
star's surface was limited to 3 nm, ensuring high meshing density
and thus good spatial sampling. The orientation dependence of the
incident E-field was accounted for by averaging the absorption
spectra of the nanostars as they were incrementally rotated by 30
degrees in the [x=y] plane, such that the orientation of the
branches relative to the z-polarized incident field became
randomized.
Example 2
Preparation of Hollow Gold Nanostars
[0114] Hollow gold nanostars were prepared using the same
seed-mediated growth method described above in Example 1 and are
shown in FIG. 9. Use of hollow gold nanoshells as the seed particle
in the method of Example 1 allows for the growth of branches while
keeping the hollow interior intact. Such particles have similar
applications to regular gold nanostars, with the added advantage of
drug or other small molecule encapsulation within the hollow
interior.
Example 3
Preparation of Magnetic Core Gold Nanostars
[0115] Gold nanostars that have superparamagnetic cores can be
synthesized by first coating superparamagnetic iron oxide (TO)
particles with a layer of gold (IO@Au), then using these IO@Au
particles as seeds in the gold-nanostar synthesis procedure
described above in Example 1. Such particles have similar
applications to regular gold nanostars, with the added advantage
that the magnetic core nanostars can be manipulated by an external
magnetic field. A schematic of this process is shown in FIG.
10.
Example 4
Imaging of Nanostars Using Two-Photon Photoluminescence (TPL)
[0116] Recently, efficient plasmon-enhanced two-photon
photoluminescence (TPL) gold nanoparticles (e.g. nanorods,
nanoshells, nanocages) have been used as a contrast agent in
several reports. On metal nanoparticles, the resonant coupling of
the plasmon band with the incident laser greatly amplifies the
nanoparticles TPL, typically increasing the two-photon action cross
sections (TPACS) of NIR-absorbing nanoparticles above those of
organic fluorophores. TPL can therefore be applied to multiphoton
microscopy, offering a convenient way to visualize NIR-absorbing
gold nanoparticles in tissue using NIR lasers.
[0117] Gold nanostars, with plasmons in the NIR, show greatly
enhanced TPL. A quadratic dependence of TPL intensity on excitation
power suggests the existence of an underlying non-linear two-photon
process on nanostars (data not shown). Such dependence was not seen
on 60 nm gold or silver spheres solution (data not shown). The TPL
excitation spectra of S20 and S30 match their plasmon spectra (see
FIG. 7), indicating that nanostars (like nanorods) enhance TPL via
plasmon coupling. Similar to previous reports, the excitation
spectra are narrower than the plasmon spectra, probably due to the
non-linear properties of TPL. Interestingly, the concentration
normalized emission intensity at 800 nm of nanostars solutions were
found to be 1.1.times.10.sup.4 greater than that of Rhodamine B,
making the TPACS of nanostars more than a million GM (data not
shown). Meanwhile, the emission intensities from each nanostars
solutions were found to be similar on the 3 different detection
channels on the microscope. The broad emission spectrum implies
that TPL from nanostars, like nanorods, may be a result of
electron-hole recombination or thermal radiation, without wishing
to be bound to any particular theory of mechanism. Because a
typical NIR dye possesses minimal TPACS, the reason for such high
TPACS from NIR-absorbing nanostars remained to be clarified.
[0118] Polymer-free nanostars with such a high TPACS can be used as
a strong contrast agent in TPL imaging in biological samples. Here,
we demonstrate TPL imaging of WheatGerm Agglutinin (WGA)
functionalized nanostars on BT549 cancer cells. A preferential
binding of WGA-coated nanostars on the cell membrane was observed
(data not shown). In contrast, PEG-coated nanostars bound poorly.
Numerous white spots representing nanostars covered the cell
membrane. The white color was a composite result of the similar
intensity on 3 different detection channels, indicating a broad TPL
emission spectrum from nanostars. The nanostars emitted strongly
without photobleaching under low laser power (4 mW), which is in
the typical working range for organic fluorophores. No signal was
observed from WGA-coated spheres under the same experimental
settings (data not shown). Interestingly, photobleaching of
nanostars was observed after several scans at higher power (>10
mW), possibly due to a structural damage of the plasmon-generating
an isotropic features. TPL imaging therefore substantiates the
biological use of these polymer-free nanostars. Exploiting its
strong TPACS, nanostars imaging and tracing without the need for
fluorophores is possible.
[0119] In conclusion, synthe plasmonically tunable gold nanostars
were synthesized which allowed for simple surface
functionalization. The synthesis was quick and polymer-free. As
branch AR increases from nanostars S5 to S30, the plasmon peaks are
shifted to the NIR region. Sharp branches interact more intensely
with NIR laser excitation, and play a key role in determining the
optical properties of the nanostars. Three dimensional models of
the nanostars were numerically solved to deduce a good agreement
between experimental and theoretical absorption spectra, with the
number of branches contributing to peak intensity and branch AR to
plasmon shift. Exploiting the high TPACS,
biomolecule-functionalized nanostars can be used as a strong
contrast agent for multiphoton microscopy as well as other NIR
imaging techniques.
[0120] Materials.
[0121] Gold(III) chloride trihydrate (HAuCl4), sodium citrate
tribasic dihydrate, L(+)ascorbic acid (AA), 4-mercaptobenzoic acid
(pMBA), silver nitrate (AgNO.sub.3), hydrochloric acid (HCl),
methanol (MeOH),
O-(3-Carboxypropyl)-O'-[2-(3-mercaptopropionylamino)-ethylJpolyethylene
glycol (Mw 5000; PEG5000), Triticum vulgaris lectin (wheat-germ
agglutinin; WGA) and rhodamine B were purchased from Sigma-Aldrich
(St. Louis, Mo.). DAP1, FM 1-43, RPMI 1640, insulin, fetal bovine
serum were purchased hom Invitrogen (Carlsbad, Calif.). 16%
paraformaldehyde was purchased from Alfa Aesar (Ward Hill, Mass.).
(Ortho-pyridyl)dis ul:fidePEG2000-Succinimidyl Ester) (Mw 2000;
OPSS-PEG2000-NHS) was purchased from Creative PEGWorks (Winston
Salem, N.C.). All chemicals were used as received. Millipore
Synergy ultrapure water (resistivity=18.2 MO em) was used in all
aqueous solutions. All glassware and stir bars were cleaned with
aqua regia solution and oven-dried before use.
[0122] Synthesis of Au Seeds.
[0123] A modified Turkevich method was used to prepare a seed
solution. Briefly, 10 ml of a 38.8 mM citrate solution was added
into 100 ml of a boiling 1 mM HAuCl.sub.4 solution under vigorous
stirring while keeping the volume roughly unchanged. After boiling
for about 30 min, the solution was cooled, filtered by a 0.22
micron nitrocellulose membrane, and kept at 4.degree. C. for
long-term storage.
[0124] Synthesis of Au Nanostars.
[0125] Nanostars were prepared by a seed-mediated growth method.
Briefly, 100 .mu.l of the above seed solution (12.+-.0.7 nm;
A.sub.520: 2.81) was added to 10 ml of 0.25 mM HAuCl.sub.4 solution
(with 10 .mu.l of 1N HCl) in a 20 ml glass vial at room temperature
under vigorous stirring. Quickly, 100 .mu.l AgNO.sub.3 at different
concentrations (5 to 30 .mu.M) and 50 .mu.l of AA (100 mM) were
added simultaneously. The solution was stirred for 30 sec as its
color rapidly turned from light red to blue or greenish-black.
Immediately afterwards, two centrifugal washes at 3000-5000 rcf
were performed in 15 ml tube to halt the nucleation. The solution
was redispersed in 2 mM pH7 citrate buffer, filtered by a 0.22
.mu.m nitrocellulose membrane, and then kept at 4.degree. C. for
long-term storage.
[0126] Instrumentation.
[0127] The structural features of the nanostars were characterized
using transmission electronic microscopy (TEM; Fei Tecnai G.sup.2
Twin, 200 kV) and analyzed using ImageJ software (National
Institute of Health). The particle hydrodynamic size distribution
and concentration were determined by nanoparticle tracking analysis
(NTA 2.1; build 0342) using NanoSight NS500 (Nanosight Ltd. UK).
Absorbance spectra were obtained using a dual-beam
spectrophotometer (Shimadzu UV-3600; Shimadzu corporation, Japan)
or a microplate reader (BMG LABTECH FLUOStar Omega; BMG LABTECH
GmBH, Germany). More than five samples were analyzed for each
synthesis condition. Morphological features were assessed from more
than 50 particles on different TEM images. Data values were
represented by mean.+-.1 standard deviation.
[0128] Modeling.
[0129] The 3-D nanostar simulations were performed using the finite
element (FEM) based Comsol Multiphysics v3.4 software package and
the RF module (Comsol, Inc. Burlington Mass., USA). A unique, 3-D
nanostar model was designed for each of the samples S5, S10, S20
and S30 using the dimensions obtained from their corresponding TEM
images (data not shown). For a particular star model, the branches
protrude normal to the core surface, but were randomly positioned
on the core such as to maximize the inter-branch distance.
[0130] The dielectric function of gold was modeled using the
Lorentz-Drude model for gold from Johnston and Christy's and the
surrounding medium was modeled as water with a refractive index
n=1.33. The computational domain was bounded by a spherical
perfectly matched layer (PML) to prevent any reflections back onto
the nanoparticle. The nanostars were excited with a z-polarized
incident plane wave of E-field amplitude 1, propagating along the
y-axis and of wavelength ranging 300 nm to 1200 nm. The
nanoparticles were meshed such that the largest mesh size on the
star's surface was limited to 3 nm, ensuring high meshing density
and thus good spatial sampling. The orientation dependence of the
incident E-field was accounted for by averaging the absorption
spectra of the nanostars as they were incrementally rotated by 30
degrees in the [x=y] plane, such that the orientation of the
branches relative to the z-polarized incident field became
randomized.
[0131] Two-Photon Microscopy Instrumentation.
[0132] The two-photon photosluminescence (TPL) images were recorded
using a commercial multiphoton microscope (Olympus FV1000, Olympus
America, Center Valley, Pa.) with 3 detection channels (420-460 nm,
495-540 mn, 575-630 nm) on photomultiplier tubes (H7422-40,
Hamamatsu, Bridgewater, N.J.). A femtosecond Ti:Sapphire laser
(Chameleon Vision II, Coherent, Santa Clara, Calif.) with tunable
range 680-1080 nm, 140 fsec pulse width and 80 MHz repetition rate
was used. The laser beam was focused through a 25 x 1.05NA
water-immersion objective (XLPL25XWMP, Olympus America, Center
Valley, Pa.). Time-correlated single photon counting fluorescence
lifetime was measured on the same microscope system using PicoHarp
300 counting electronics (PicoQuant GmBH, Berlin, Germany).
[0133] Two-Photon Action Cross Section (TPACS) Measurement of
Nanostars.
[0134] The TPACS of gold nanostars (0.1 nM in 2 mM citrate buffer)
were measured by comparing the two-photon photoluminescence of
nanostars with fluorescence of Rhodamine B (100 nM in pure MeOH) at
800 nm excitation. The TPACS has a unit of cm.sup.4s, where 1
Goeppert-Mayer unit (GM) represents 10.sup.-50 cm.sup.4s/photon.
The excitation power was 2 mW.
[0135] Luminescence/fluorescence intensity was integrated over an
image area of 250.times.250 .mu.m.sup.2 under 10 .mu.s/pixel and
256.times.256 resolution. The sample solution was placed on a
depression slide covered by a coverslip during imaging.
[0136] Cell Culture;
[0137] The BT549 cancer cells were a gift from Victoria Seewaldt's
lab. Cells were incubated in RPMI 1640 culturing media, containing
10% of fetal bovine serum and 0.023 U/ml of insulin, in an
incubator with a humidified atmosphere (5% CO.sub.2). Cells in
exponential growth phase were used in experiments.
[0138] TPL Cellular Imaging.
[0139] Nanostars were conjugated with WGA using a
hetero-bifunctional crosslinker OPSS-PEG.sub.2000-NHS based on a
previous protocol. WGA has high affinity to glycoproteins and
glycolipids on the cell membrane therefore WGA-conjugated nanostars
can be seen on cell surface. Nanostars conjugated with PEG.sub.5000
were used as control. Functionalized nanostars were washed and
resuspend to a final concentration 0.1 nM in PBS. Paraformaldehyde
(4%, 10 min) fixed cells, pretreated with FBS blocking for 3 min
and PBS washes, underwent 10 min incubation with WGA-nanostars or
PEG-nanostars followed by PBS washes. Counterstain with Hoescht
33342 and FM 1-43 FX were performed according to the company's
protocols. Two-photon imaging was done under 1% transmission, 10
.mu.s/pixel and 512.times.512 resolution with 4-frame Kalman
averaging. All 3 channels were set to 550 gain (for
WGA-nanostar)/600 gain (for PEG-nanostar) and 8 offset.
Example 5
Photothermal Properties of Nanostars
[0140] Selective localized cancer treatment can be achieved when
the laser irradiates a tumor concentrated with nanostars that can
act as an efficient photothermal transducer. The plasmonic gold
nanostars of the present disclosure offer such a unique capability
to transduce photon energy to heat when excited in the near
infrared (NIR) tissue therapeutic window. The gold nanostars, with
their small core size and multiple long thin branches, exhibit high
absorption cross sections that are tunable in the NIR region with
relative low scattering effect, rendering them efficient
photothermal transducers. Here, photothermal ablation was
demonstrated in SKBR3 breast cancer cells incubated with nanostars
synthesized according to the method described in Example 1
undergoing laser irradiation of different durations. Also, a
regional hyperthermia to 45.degree. C. was observed when
irradiating a region injected with the nanostars. The results of
plasmon-enhanced localized hyperthermia have illustrated the
usefulness of gold nanostar as an efficient photothermal agent in
cancer therapy.
[0141] Materials.
[0142] Gold(III) chloride trihydrate (HAuCl4), sodium citrate
tribasic dehydrate (Na.sub.3Cit), L(+)-ascorbic acid (AA), silver
nitrate (AgNO3),
0-(3-Carboxypropyl)-0'-[2-(3-mercaptopropionylamino)ethyl)-polyethylene
glycol (Mw 5000; SHPEG5000), were purchased from Sigma-Aldrich (St.
Louis, Mo.). McCoy 5A, fetal bovine serum, Hoescht 33342, FM 1-43FX
were purchased from Invitrogen (Carlsbad, Calif.). All chemicals
were used as received. Millipore Synergy ultrapure water
(resistivity=18.2 M.OMEGA. cm) was used in all aqueous solutions.
All glassware and stir bars were cleaned with aqua regia solution
and oven-dried before use.
[0143] Gold Nanostars Synthesis.
[0144] Gold nanostars were synthesized as described in Example 1
herein above.
[0145] Gold Nanostars Characterization.
[0146] Transmission electronic microscopy (TEM; Fei Tecnai G2 Twin,
200 kV) was used for structural analysis. The particle hydrodynamic
size distribution, concentration, and s-potential were determined
by nanoparticle tracking analysis (NTA 2.1; build 0342) using
NanoSight JSSOO (Nanosight Ltd. UK). A UV-VIS spectrophotometer
(Shimadzu UV-3600; Shimadzu corporation, Japan) was used to collect
the extinction spectrum.
[0147] Cell Culture and Incubation with Gold Nanostars.
[0148] The SKBR3 breast cancer cells were cultured in McCoy 5A
culturing media (10% fetal bovine serum) in an incubator with a
humidified atmosphere (5% CO2) according to the ATCC's protocol.
Cells in exponential growth phase were used in experiments. The
cells were seeded into 35 mm petri dishes for more than 2 days
until .about.80-90% confluency. Unlabeled nanostars (0.3 nM
particle concentration) were suspended and sonicated in the same
growth media immediately before use. Nanostars functionalized with
SHPEG5ooo 5 .mu.M were washed and resuspended (0.3 nM) in growth
media. Then, cells were incubated 1 hour with 1 ml of (1)
unmodified nanostars in media, (2) PEGylated nanostars in media and
(3) media alone. Before the PTT, cells were washed by PBS twice and
replaced with new media. Another set of cells were fixed by 4%
paraformaldehyde and imaged under multiphoton microscopy (Olympus
FV 1000, Olympus America, Center Valley, Pa.) to confirm the
presence of nanostars in cells. Cells were stained with Hoescht
33342 (2 .mu.g/ml in PBS) and FM 1-43 FX (4 .mu.g/ml in PBS) 30 min
prior to imaging.
[0149] Phorothermal Therapy and Viability Staining.
[0150] On a 37.degree. C. heating stage, cells with 90% confluency
were exposed to 980 nm diode laser irradiation (15 W/cm.sup.2, spot
size 8 mm.sup.2) for 1, 3, and 5 min. Both nanostar-treated and
untreated samples receiving no laser irradiation were used as
control. After 1 day, cells were examined by a live-cell staining
using Fluorescein diacetate (FDA; 1 .mu.g/ml in PBS, incubated for
30 min) under a fluorescence microscope. Non-fluorescent FDA is
converted to green fluorescent fluorescein by esterase in living
cells.
[0151] In Vivo Photothermal Treatment.
[0152] A nude athymic mouse was anesthetized by ketamine and kept
warm on a 37.degree. C. heating stage. A 50 .mu.l of PEGylated gold
nanostars solution (6 nM in PBS) was injected subcutaneously on one
side of thigh with saline injection on the other thigh as control.
The injection sites were irradiated (980 nm, 0.56 W/cm2, spot size
1 cm2) for 5 min. The temperature was measured by a needle
thermocoupler before and after the laser irradiation. The animal
experiments were conducted in compliance with the guidelines for
the care and use of research animals by IACUC.
[0153] In this study, plasmon tunable gold nanostars were employed
as a new photothermal transducer for hyperthermic therapy. Based on
theoretical calculations, at the plasmon peak, nanostars achieve an
absorption to scattering cross section ratio greater than the
nanorods and nanoshells. In vitro, nanostars locate predonantely in
the cytoplasm, as investigated by the two-photon photoluminescence
imaging. The photothermal response was maximal when both NIR laser
excitation and nanostars incubation were combined but only minimal
when separated. The localized cell death intensified after longer
irradiation suggesting a laser dose dependency.
[0154] Nanostars were synthesized with size around 60-70 nm. The
synthesis was simple without the use of polymer. TEM showed
star-shaped particles with 8-10 protruding branches on a small
core. Some branches even had additional small branches on them.
Nanostars exhibit a broad plasmon spectrum, which peaks at 890 nm
with a molar absorptivity of 6.7.times.10.sup.9 M-1 cm-1 The
hydrodynamic size was 70.+-.32 nm and the .zeta.-potential was
-31.3 mV by nanoparticle tracking analysis. Upon addition of the
serum-containing culturing media, the plasmon peak redshifted,
suggesting possible aggregation.
[0155] The nanostars efficiently absorb NIR energy. The modeled
nanostar had plasmon peaks at 960 nm with the absorption to
scattering ratio of nanostar 9.2. The theoretical simulation
results indicate that nanostars predominately absorb rather than
scatter the incident photoenergy in the simulation. It was
determined that the branches aspect ratio, but not the core size or
branch number, contributes the major role for the red-shifted
plasmon. Here, the modeled nanostars have the aspect ratio changed
from 1.46 to 2.1 with branch number increased from 8 to 10 and core
size decreased from 23.7 to 21.7. At their plasmon peaks, the
absorption to scattering ratios (C.sub.Abs/C.sub.Sca) of nanostar A
and B are 10.7 and 9.2, respectively (Table 2).
TABLE-US-00002 TABLE 2 Calculated cross section comparison of
aspherical gold nanoparticles (AuNPs) C.sub.Abs C.sub.Sca
(10.sup.-15 m.sup.-2) (10.sup.-15 m.sup.-2) C.sub.Abs/C.sub.Sca
Nanorods, width: 17.9, R: 3.9, 16.9 5.0 3.38 at 820 nm Nanoshells,
R1/R2: 60/70 nm, 50.9 32.5 1.57 at 892 nm Nanocages, core: 30 nm,
7.3 0.8 9.1 at 825 nm Nanostars A, at 810 nm 11.4 1.07 10.7
Nanostars B, at 960 nm 14.0 1.52 9.2
[0156] Compared to other classes of aspherical AuNPs, nanostars
have comparable ratio to nanocages, but much higher ratio than
norods and nanoshells. Because the scattering is proportional to
the particle size, the nanocages of 30-nm core typically have
smaller scattering cross section. For nanostars, although the
tip-to-tip diameter is 70-nm, the core size is small and branches
are thin. The scattering thus is smaller than 70-nm of nanorods and
nanoshell. Since it is desirable to have low scattering and high
absorption, nanostars can be a better candidate agent for
photothermal therapy. Furthermore, nanostars efficiently absorb NIR
energy in the therapeutic window region. In a solution filled with
1 nM and 0.1 nM nanostars excited by 980 nm CW laser of 1.2 W, the
temperature rose to 80.degree. C. and 60.degree. C. respectively in
5 min (data not shown). Meanwhile, the 2-mM citrate solution only
rose to 40.degree. C. under the same laser excitation. To avoid
thermal damage from the 980-nm laser itself, the possible laser
excitation time was set to less than 5 min.
[0157] Prior to PTT experiments, the uptake of nanostars was
investigated by two-photon luminescence (TPL) microscopy. Because
nanostars possess a high two-photon action cross section, the
presence of nanostars in cells can be easily detected under a
commercial multi-photon microscopy. After one-hour of incubation,
bright nanostars signal could be seen only in cells receiving
media-treated nanostars (data not shown). Even without any surface
functionalization, the nanostars located predominately in the
cytoplasm but not the nucleus displaying a ring pattern on the
image. Cells receiving no nanostars showed only weak
autofluorescence (data not shown).
[0158] In one experiment, separated wells filled with 3 nM and 0.3
nM PEGylated nanostars 200 .mu.l were irradiated by 980 nm CW diode
laser (15 W/cm.sup.2, 0.08 cm.sup.2); the temperature rose to
80.degree. C. and 60.degree. C. respectively in 5 min (data not
shown). The nanostar-free Na.sub.3Cit buffer rose to 40.degree. C.
under the same laser irradiation, suggesting a relatively low but
potential risk of thermal damage under this power density. To avoid
thermal damage from the 980 nm laser itself, the laser irradiation
time was set to maximal of 5 minutes. Prior to PTT experiments, the
uptake of nanostars was investigated by two-photon luminescence
(TPL) microscopy. After one-hour of incubation, bright dots
representing nanostars could be seen only in cells incubated with
nanostars but not PEGylated nanostars (data not shown). These
unlabeled nanostars located predominately in the cytoplasm
displaying a ring pattern on the image. Meanwhile, cells incubated
with no nanostars showed only dye signals (data not shown). The
photothermal response on SKBR3 cells was measured using 3 different
laser treatment durations (data not shown). With the presence of
nanostars, cancer cells were killed after 3 or 5 min but were not
visibly harmed after 1 min of laser irradiation. Once dead, cells
in the treated region detached before imaging. The photothermal
response was higher at 5 min than at 3 min reflecting the laser
dose dependency. Cells without nanostars were viable until 5
minutes of irradiation, when a small region of cells began to die.
This was probably due to the temperature elevation from the laser
irradiation itself, although the ablated area was much smaller than
cells treated with nanostars and laser combined. Meanwhile, cells
incubated with nanostars but not treated with the laser maintained
their viability. Thus, no apparent cytotoxicity was observed from
nanostars during the study period.
[0159] The photothermal response in vivo was examined by
irradiating a region injected subcutaneously with PEGylated
nanostars (data not shown). A bluish bulging discoloration was the
area of nanostars injection. Saline injection resulted in quick
dispersion with no visible elevation. The laser spot size was
adjusted to 1 cm.sup.2 to cover the injection site. In 5 min, the
temperature rose from 32.8 to 45.degree. C. For saline control, the
temperature increased to 39.5.degree. C.
[0160] The effectiveness of nanostars as photothermal transducers
has been demonstrated by the study described herein. First, the
synthesis of NIR absorbing nanostars can be performed in only
minutes, which is much quicker than other classes of aspherical
AuNPs that can take more than hours to synthesize. Second, since it
is desirable to have low scattering and high absorption, nanostars
can be a better agent for photothermal therapy. From the simulation
results, nanostars were shown to have compatable Abs/Sca ratio to
nanocages, but much higher ratio than nanorods and nanoshells.
Because the scattering is proportional to the particle size, the
nanocages of 30-nm core typically have smaller scattering cross
section. For nanostars, although the tip-to-lip diameter is 70-nm,
the core size is small and branches are thin. Thus, the scattering
for nanostars of 70-nm is smaller than that of nanorods and
nanoshells of 70-nm.
[0161] The photothermal response is determined by the both
nanostars (plasmon position, particle concentration) and laser
(wavelength, irradiation power and duration). The strong and broad
plasmon spectrum of nanostars matches well the wavelength of the
NIR laser. Within 5 minutes of laser irradiation, the temperature
of 3 nM and 0.3 nM nanostars-containing solutions increased by 56
and 38.degree. C., respectively, while Na.sub.3Cit buffer solution
only increased by 18.degree. C. Although the laser wavelength used
in the study is beyond the range of tissue optical window due to
the availability of the laser, the difference in temperature
profile clearly indicates the photothermal transduction effect of
nanostars. The photothermal ablation using nanostars was
demonstrated on SKBR3 breast cancer cells.
[0162] Nonspecific cellular uptake of unlabeled nanostars showed a
concentration dependent manner (data not shown).
Na.sub.3Cit-stabilized nanostars, when treated with FBS-containing
media, resulted in passive adsorption of serum protein onto the
particle surface. The non-specific interaction between serum
proteins and cell membrane might facilitate the uptake of
nanostars. Although nanostars were aggregated, the clustering might
also increase the uptake. When combining a localized irradiation to
cells accumulated with nanostars, a confined region of killing can
be observed within 3 minutes, while laser irradiation or nanostars
incubation alone generated no killing effect. With longer
irradiation time, the photothermal ablation effect became more
prominent. Unfortunately, due to higher water absorption at 980 nm,
a small area of cells was non-specifically ablated after longer
irradiation.
[0163] The photothermal response in vivo was demonstrated on an
athymic nude mouse. Based on the ANSI regulation, the maximal
permissible exposure to skin at 980 nm was 0.73 W/cm.sup.2. Here, a
power density of 0.56 W/cm.sup.2 was applied to the area filled
with nanostars in the subcutaneous layer. At this power density, a
12.degree. C. of temperature increase was observed after 5 minutes
of irradiation. Slight increase in injection area can be seen due
to diffusion of nanoparticles. After the irradiation, a visible
inflammation was noticed on the margin of the injection site
probably as a result of thermal injury. In contrast, irradiating
area of saline injection showed only 7.degree. C. of temperature
increase due to slightly high tissue absorption at 980 mn. Still no
visible skin damage was seen. Further optimization to increase
nanostars targeted binding, to improve nanostars stability in
physiological environment, and to reduce non-specific laser heating
would further enhance the selectivity and efficacy of photothermal
killing. In this study, we employed gold nanostars as a new
photothermal transducer for hyperthermic therapy. The nanostars
synthesis is simple and quick. Based on theoretical calculations,
at the plasmon peak nanostars exhibit an Abs/Sca cross section
ratio greater than nanorods and nanosbells. In vitro, unlabeled
nanostars locate predominately in the cytoplasm, as investigated by
the two-photon photoluminescence imaging. The photothermal response
was maximal when both NIR laser irradiation and nanoslars
incubation were combined but only minimal when separated. The
localized cell death intensified after longer irradiation
suggesting a laser dose dependency. In vivo, a localized
hyperthermia was achieved by irradiating an area containing
subcutaneous injection of PEGylated nanostars but not saline.
According to the methods of the present disclosure, size and
plasmonic properties of nanostars can be optimized for photothermal
response using laser in the tissue optical window.
Example 6
Silica-Coated Gold Nanostars for Combined SERS Detection and
Photodynamic Treatment (PDT)
[0164] The synthesis of SERS-tagged gold nanostars coated with a
silica shell encapsulating methylene blue (MB) is presented as a
construct for combined PDT and SERS imaging. The surface plasmon
band of the nanostars and the absorption band of the Raman reporter
used fall within the NTR region. This is ideal for in vivo imaging
since tissue absorption is minimal in this range, increasing the
penetration depth of the excitation source and the efficiency of
Raman scattering collection. Use of an excitation source that
overlaps with the absorption of the Raman reporter allows for
surface-enhanced resonance Raman scattering (SERRS), further
enhancing the efficiency of Raman scattering by a few orders of
magnitude. It is demonstrated herein that the core-shell particles
with encapsulated MB show enhanced singlet oxygen generation as
compared to core-shell particles grown without MB. Optical
characterization of the nanocomposites was performed by Raman,
fluorescence, and Vis-NIR absorption spectroscopies.
[0165] Materials.
[0166] Gold(111) chloride trihydrate (HAuCl.sub.4.3H.sub.2O),
trisodium citrate dehydrate
(C.sub.6H.sub.5O.sub.7Na.sub.3.2H.sub.20), 1N HCl, tetraethyl
orthosilicate (TEOS),
O-[2-(3-Mercaptopropionylamino)ethyl]-O'-methylpolyethylene glycol
(mPEG-SH, MW 5 k), methylene blue hydrate (MB),
3,3'-Diethylthiatricarbocyanine iodide (OTIC), and methanol were
purchased from Sigma-Aldrich at the highest purity grade available.
Silver nitrate (AgNO.sub.3, 99.995%) was supplied by Alfa Aesar.
Pure-grade ethanol and NH.sub.4OH (29.5%) were obtained through
VWR. Ultrapure water (18 M.OMEGA. cm-1) was used in all
preparations. The Singlet Oxygen Sensor Green (SOSG) reagent was
procured from Invitrogen.
[0167] Nanostar Synthesis.
[0168] Briefly, 12-nm gold seeds were synthesized by reduction of
gold(III) with citrate. Nanostars were grown from the seed by
reduction of gold(III) chloride with ascorbic acid in the presence
of silver nitrate under acidic conditions. The stock concentration
of particles is approximately 0.1 nM, as determined by Nanoparticle
Tracking Analysis (NTA).
[0169] SERS-Tagging.
[0170] Freshly synthesized nanostars (10 mL) were capped with 5
.mu.M mPEG-SH under gentle stirring for 15 minutes. The PEGylated
particles were then centrifuged (10 k rcf, 15 minutes) twice at
4.degree. C. to remove excess PEG and redispersed in water. To this
solution, 5 .mu.M DTTC in methanol was added and allowed to stir
overnight. The DTTC-tagged particles were centrifuged (5 k rcf, 15
minutes) twice at 4.degree. C. and resuspended into 2.3 mL of
ethanol.
[0171] Methylene Blue Encapsulation by Silica Coating.
[0172] A modified Stober method was used for formation of the
silica shell. Under gentle stirring, 2.25 mL of the nanostars in
ethanol was added to a solution containing 2.0 mL of water and 6.8
mL ethanol. Methylene blue (final concentration 5 .mu.M) in ethanol
and 160 .mu.L of NH.sub.4OH were added to the mixture. Silica
coating was initiated by the addition of 30 .mu.L 10% TEOS in
ethanol, and the reaction was allowed to proceed for three hours.
The particles were then washed until no MB absorption could be
detected from the supernatant (typically 2-3 times) at 4.degree. C.
by centrifugation (3.5 k rcf, 15 minutes) and redispersed into 5 mL
of water.
[0173] Characterization.
[0174] Raman spectra with 785-nm excitation (25 m W) were recorded
on a Jobin Yvon LabRAM ARAMIS system using a 1200-g mm-1 grating.
Fluorescence and Raman spectra with 633-nm (8 mW, 10% laser power
for fluorescence, 100% for Raman) excitation were recorded on a
Renishaw inVia Raman microscope using an 1800-g mm-1 grating.
Transmission electron microscopy (TEM) was performed on a FEI
Tecnai G.sup.2 Twin transmission electron microscope with an
accelerating voltage of 160 kV. Vis-NIR spectra were acquired on a
Shimadzu UV-3600. Particle concentrations were measured by NTA with
a NanoSight NS500.
[0175] Singlet Oxygen Generation.
[0176] The fluorescent probe, SOSG, was used to indirectly measure
singlet oxygen generation from the particles. On a 96-well plate,
90 .mu.L of the coated particles were mixed with 10 .mu.L of
.about.100 .mu.M SOSG in methanol. The sample was excited using
63-nm laser light focused into the solution with a 10.times.
objective. Laser power was 8 mW at the sample. Fluorescence
intensity was measured with a BMG LABTECH FLUOstar Omega using an
excitation filter of 500.+-.10 nm and emission filter of 530.+-.10
nm.
[0177] The use of PEG as a surfactant has been shown to increase
the colloidal stability of gold nanoparticles enough to allow for
redispersion into ethanol without aggregation. The PEG molecules
have also been shown to be effective in facilitating the
condensation of silica onto nanoparticles, creating a silica shell.
Here PEG is utilized in silica coating to encapsulate MB in a
mesoporous shell around gold nanostars of the present disclosure.
Previous work has shown that MB is loaded into the silica matrix
when present in solution during silica condensation using the
Stober method. Unexpectedly, the capping of the gold nanostars of
the present invention with PEG did not seem to interfere with the
ability to tag them with the Raman reporter DTTC. It is presumed
that the sulfur groups in the DTTC molecule aid in adsorption to
the gold surface. Upon laser excitation at 785 nm, the DTTC-tagged
particles showed no fluorescence signal, indicating that the dye is
located at or near the gold surface, resulting in quenching. A
strong SERRS signal was observed at 785 nm from silica encapsulated
MB DTTC-tagged particles (AuNS-DTTC@SiO.sub.2-MB), while the gold
nanostars with silica encapsulated MB without DTTC
(AuNS@SiO.sub.2-MB) exhibited no appreciable signal (data not
shown). Raman spectra of AuNS-DTTC @ SiO.sub.2 particles without MB
showed a much weaker signal when excited at 633 nm (data not
shown). This is likely because the excitation is no longer resonant
with the absorption band of DTTC or the Plasmon band of the
nanostars. Silica coating of the AuNSs gave the expected red-shift
in the Vis-NIR absorption spectrum due to an increase in the local
refractive index around the nanoparticles. There appeared to be a
small shoulder around 680 nm for the silica coated particles that
may be attributed to the encapsulated MB. FIG. 11 shows a TEM image
of the silica coated particles. Upon laser excitation at 633 nm,
strong fluorescence is observed from the silica coated particles
containing MB (FIG. 12). The AuNS-DTTC@SiO.sub.2-MB show a blue
shift of .about.3 nm in the fluorescence spectrum compared to the
AuNS-DTTC@SiO.sub.2 sample spiked with 0.5 .mu.M MB. Previous
studies have suggested that caging and confinement effects of the
silica matrix on an embedded dye can cause a blue shift in the
fluorescence emission.
[0178] A centrifugation study was performed to examine the leaching
behavior of MB from the silica matrix. Solutions of particles were
acidified to pH 3 with HCl to limit the adsorption of MB onto the
outer surface of the silica shell. The fluorescence emission of
as-prepared AuNS-DTTC @SiO.sub.2-MB was measured after varying
numbers of washes using 633 nm excitation. For each wash, samples
were centrifuged at 3.5 k rcf for 10 minutes, decanted, then
redispersed in pH 3 water/HCl. The same measurements were performed
on AuNS-DTTC@SiO.sub.2 spiked with 0.5 .mu.M MB, giving a similar
initial fluorescence intensity as the AuNS-DTTC@SiO.sub.2-MB (see
FIG. 12). Measured intensity for the MB encapsulated and MB spiked
particles was normalized by the initial intensity for each sample,
respectively. It is shown that excessive washing can leech MB out
of the silica matrix, although slower than MB adsorbed to the
silica surface (data not shown).
[0179] Singlet oxygen generation from the particles was measured
indirectly with the fluorescent probe, SOSG. Upon reaction with
singlet oxygen, SOSG becomes fluorescent with excitation at a lamda
max of 504 and emission at a lamda max of 525 nm. Singlet oxygen
generation from AuNS-DTTC@SiO.sub.2-MB was compared with
AuNS-DTTC@SiO.sub.2 (data not shown). The measured fluorescence
intensities at each time point were nonnalized to the initial
fluorescence of the sample. A significant increase in the amount of
singlet oxygen generation was observed from the MB embedded
particles.
[0180] Described above are SERS-tagged nanocomposites possessing a
combined capability for SERS detection and singlet oxygen
generation for PDT. This work has demonstrated the strong SERRS
signal from DTTC-tagged nanostars at 785 nm laser excitation.
Encapsulation of MB photosensitizing drug into a silica shell
around the nanostars shows enhanced singlet oxygen generation upon
laser excitation at 633 nm compared to silica coated nanostars
without MB. These multimodal nanoprobes can be useful for
therapeutic applications, diagnostics, and integrating SERS imaging
and PDT.
Example 7
SERS of Nanostars
[0181] Monodisperse 50-nm gold nanostars (AuNSs) were synthesized
according to the method described in Example 1, which were easily
labeled with SERS active dyes without the need for removal of
surface polymer on the nanoparticles. Spherical silver (AgNPs) and
gold (AuNPs) nanoparticles of roughly 50-nm in diameter were also
prepared. For spherical silver nanoparticles, because both standard
citrate and hydroxylamine reduction methods produced a wide size
distribution. Seed-mediated methods of synthesis were utilized to
create monodisperse 50-nm AgNPs. This AgNP, however, was sensitive
to oxidation; SERS dyes addition to a 3-day old AgNP sample had
much less SERS intensity than the fresh-made AgNP. Because of this,
all AgNP studied were prepared fresh. For spherical gold
nanoparticles (AuNPs), the modified Turkevich citrate reduction
method produced slightly oval-shaped, monodisperse 50-nm AuNPs.
Both gold nanostars and spheres did not show significant oxidation
effect.
[0182] The extinction spectrum maximum of the three different
nanoparticles ranges from 400-800 nm based on their composition and
geometry (FIG. 13). Previously, it has been shown on simulation
that nanostars' plasmon peak and intensity are determined by the
branch aspect ratio and branch number/length, respectively. Because
of the heterogeneous branch morphology, nanostar ensembles probably
enable a wider range of LSPR modes, which explains the broadening
of the extinction spectra. Upon the addition of 1 .mu.M 4-MBA,
which has less than the full surface coverage (20.about.40%) on
nanoparticles and did not induce apparent aggregation, a slight
spectral red-shift was observed. The plasmon red-shifted more on
AgNP (4 nm) than on AuNP (1 nm); this probably reflects the
different thiol binding efficiency between silver (59 kcal/mol) and
gold (45 kcal/mol). For nanostars, even though their branches
contain a small amount of silver, enhanced thiol binding does not
explain the red-shift difference between nanostars (15 nm) and AgNP
(4 nm). A previous report found on simulation a remarkable plasmon
red-shift on nanostars when the surrounding refractive index
increases. It is therefore very likely that the plasmon from the
branches is more sensitive to the refractive index change than the
plasmon from the spheres, hence a significant plasmon red-shift on
nanostars.
[0183] FIG. 13 shows the non-resonance SERS comparison of
nanostars, AgNP and AuNP, with the most prominent peaks being the
Stokes features appearing at 1013 cm.sup.-1 and 1078 cm.sup.-1,
which are assigned to the C--O stretching mode of MeOH and ring
breathing mode of the 4-MBA. It is noteworthy that no NaCl was
added to avoid nanoparticle aggregation. Although there was no
strong evidence of aggregation on UVVIS, nanoparticle tracking
analysis still showed a small amount of larger particles on all
samples after the addition of 4-MBA.
[0184] Because adding 4-MBA generated the smallest size change when
compared to adding 4-aminothiophenol, thiophenol, and
4-methylbenzinethiol, it is believed that nanoparticle ensembles
labeled with 4-MBA were the closest to their non-aggregated state.
This allows for SERS measurement to reflect the realistic EM
enhancement on nanoparticle ensembles. Although this is not a
single particle SERS study, the ensemble-averaged signal still
provides crucial information for SERS comparison between different
compositions and geometries. At 785 nm, nanostars exhibited a
slightly greater enhancement than AgNP but significantly
outperformed AuNP (data not shown). The EF of isolated AuNP was not
visible in our setup; an EF of lower than 4 orders of magnitude on
isolated AuNP was previously reported. Nanostars, with the presence
of multiple hot spots on the branches, have at least comparable EF
to AgNP, which is typically 2 to 3 orders of magnitude stronger
than AuNP. Being able to achieve a similar EF to AgNP without the
toxicity of silver, nanostars are particularly advantageous due to
the superior biocompatibility of gold. Moreover, because
aggregation is generally hard to control and irreproducible, the
advantage of having strong SERS without the need to form aggregates
make gold nanostars a useful SERS contrast agent.
[0185] FIG. 14 illustrates a laser wavelength dependence of SERS EF
on both AgNP and nanostars. Generally, maximal enhancement occurs
at excitation near the LSPR maximum. With AgNP, which has a plasmon
maximum located near 405 nm, a 2-fold stronger EF was obtained from
785 nm than 633 nm excitation, suggesting a possible minute
aggregation dominating the overall SERS response. Such aggregation
was barely detectable under UVVIS but was only slightly visible
under Nanosight. On nanostars, more than 10-fold stronger EF was
observed under excitation from 785 nm than from 633 nm. Stronger
SERS response has previously been reported under 785 nm than at
shorter wavelength. Under 633 nm excitation, the ensemble-averaged
nanostars SERS EF (0.3.times.10.sup.5) is lower than previously
reported (2-5.7.times.10.sup.5), probably due to different
dyes/nanostars concentrations or NaCl used.
[0186] Under 785 nm excitation, the ensemble-averaged nanostars
SERS EF is around 4.times.10.sup.5, which is slightly higher than
previous results of (1.times.10.sup.5) and (2-5.times.10.sup.3).
Having sharper branches and no polymer coating on the nanostars
provided herein might contribute the slight EF improvement.
However, it is noteworthy that the calculated SERS EF decreases
with increasing analyte concentrations (data not shown). At lower
analyte concentrations it is likely that most analyte molecules
attach to the outermost part of the nanostars (i.e. the tip) where
the strongest EM field locates. At higher analyte concentration,
molecules may also bind to inner regions (e.g. the shaft, trough);
the higher EM enhancement at the tips is then averaged out by
fields from the shaft or other regions. Such phenomenon is more
apparent under 785 nm excitation, which is in resonance with the
branch plasmon. Therefore, a near 10-fold stronger EF was found at
the lower concentration than the higher one. This is in agreement
with the "lightning rod" effect demonstrating the role of branch
tip as hot spot. Without wishing to be bound by any particular
theory of mechanism, in short, a combination of LSPR matching the
excitation laser and presence of multiple hot spots on nanostars
may explain their strong SERS enhancement.
[0187] However, although nano stars have slightly higher EF than
gold nanorod ensembles (10.sup.4-10.sup.5), its EF is still lower
than that from gold dimers (10.sup.7-10.sup.8) or clustered
patterns (10.sup.8-10.sup.10). With more branches per nanoparticle,
it is reasonable that nanostars have several fold higher
enhancement than nanorods. However, the EF from sharp protrusion
may be less than from a coupled configuration. Dimers, despite
their higher EF, remain difficult to fabricate uniformly.
Film-based clustered patterns cannot be applied easily to
biological systems as well. Although nanostars coupled to a gold
film produced SERS EF of 10.sup.10, enhancement that high could not
be reached even upon NaCl induced aggregation in solution.
[0188] It is noteworthy that the ensemble-averaged SERS increment
upon aggregation was much less profound from nanostars than from
spheres. Instead of a branch facing another branch, aggregated
nanostars probably have branches collapsing into each other, hence
breaking the hot spots on the branches. It is likely that nanostar
aggregates produce much less EM enhancement than sphere aggregates
due to the possible plasmon deactivation from interaction between
tips with different geometry and orientation. Although aggregates
may produce strong SERS, keep in mind that the reproducible
formation of, as well as access to, hotspots in nanoparticle dimers
or aggregates is limited. Controlled aggregation in ensemble is
appealing but still remains a difficult task with various yields.
In contrast, nanostars possess a high EF as non-aggregated
monodisperse entities, and therefore do not suffer from the
non-linear enhancement effect due to aggregation. Until a more
reliable method is developed for the formation of reproducible and
accessible hot spots, nanostars may nonetheless be one of the most
sensitive and controllable SERS platforms in solution.
[0189] To fabricate a strong SERS probe for bioapplications,
several dyes were investigated. Non-thiolated dyes (e.g. rhodamine,
crystal violet) were found unable to remain on the nanoparticle
surface during the silica coating process. Thiolated, thiocyanated,
thionine dyes, such as 4-MBA, fluorescein isothiocyanate, methylene
blue, all sustained the silica coating process but the resulting
SERS signal under 785 nm excitation remains insufficient, requiring
long integration time for adequate signal-to-noise ratio. One
exception is 3,3'-diethylthiatricarbocyanine iodide (DTTC), which
is a NIR dye that has two cyanine groups that will facilitate its
anchoring onto the gold surface. DTTC is also resonant with the 785
nm excitation laser, creating a stronger resonance Raman for use in
SERS detection. Upon silica coating, the hydrodynamic size of the
SERS probes used in this study were around 110 nm (data not
shown).
[0190] FIG. 15 shows the SERS spectra of silica-coated DTTC-encoded
SERS probe in different compartments of cells with short
integration time (10 sec). Similar strategies have been applied for
SERS mapping in cells or tissues previously. In our study,
two-photon microscopy disclosed that SERS probes were accumulated
mostly in the cytoplasm with minimal intranuclear accumulation
after 24 hours of probe incubation (FIG. 15). SERS signals were
found to be the greatest in the cytoplasm region but remained
one-fifth as strong in the nuclear region. The discrepancy between
the two-photon image (minimal intranuclear nanostars) and SERS
spectra could be the fact that due to a low axial resolution (from
the large axial optical probe volume), the SERS spectra collected
in the nuclear region may be confounded by signals from the
cytoplasm above or underneath the nucleus. Further use of
multifunctional SERS probes by incorporating other modalities (e.g.
photodynamic therapy, photothermal therapy, MRI contrast) may bring
forth promises for molecular imaging and cancer therapy.
[0191] Chemicals.
[0192] Gold (III) chloride trihydrate (HAuCl.sub.4), sodium citrate
tribasic dihydrate (Na.sub.3Cit), L(+)-ascorbic acid (AA), silver
nitrate (AgNO.sub.3), hydrochloric acid (HCl),
0-[2-(3-mercaptopropionylamino)ethyl]-O'-methylpolyethylene glycol
(M.sub.w 5,000; SHPEG.sub.5k), 4-mercaptobenzoic acid (4-MBA),
ethanol (EtOH), methanol (MeOH), tetraethyl orthosilicate (TEOS),
3,3'-diethylthiatricarbocyanine iodide (DTTC) were purchased from
Sigma-Aldrich (St. Louis, Mo.). All chemicals were used as
received. Millipore Synergy ultrapure water (DI) of
resistivity=18.2 M.OMEGA. cm was used in all aqueous solutions. All
glassware and stir bars were cleaned with aqua regia solution and
oven-dried before use. (Caution: aqua regia is extremely dangerous.
Please use it with extra caution.)
[0193] Gold Seed Synthesis.
[0194] Gold seeds were made by adding 15 ml of 1% (w/v) Na.sub.3Cit
solution into 100 ml of boiling 1 mM HAuCl.sub.4 solution under
vigorous stirring. After 30 min, the solution was cooled, filtered
by a 0.22 .mu.m nitrocellulose membrane, and kept at 4.degree. C.
for long-term storage.
[0195] Gold Spheres (50 nm) Synthesis.
[0196] Gold spheres were made by adding 0.8 ml of 1% (w/v)
Na.sub.3Cit solution into 100 ml of boiling 0.25 mM HAuCl.sub.4
solution under vigorous stirring. After 30 min, the solution was
cooled, filtered by a 0.22 .mu.m nitrocellulose membrane and stored
at 4.degree. C. for long-term storage. Before use, the solution
underwent centrifugal wash (3000.times.g for 15 min) once and was
resuspended to 0.1 nM.
[0197] Silver Spheres (50 nm) Synthesis.
[0198] Silver spheres were synthesized using a seed-mediated method
modified from the nanostars synthesis method below. In 10 ml of 100
mM AgNO.sub.3 solution, 100 .mu.l of the citrate gold seed solution
was added under room temperature. Immediately afterwards, a mixture
of 50 .mu.l of 100 mM AA and 10 .mu.l of HCl was administered. One
hour later, the solution underwent centrifugal wash (3000.times.g
for 15 min) once and was resuspended to 0.1 nM. The Ag spheres were
used within a day to reduce the detrimental effect from surface
oxidation.
[0199] Nanostars Synthesis.
[0200] Gold nanostars were synthesized using a seed-mediated
method. Detailed synthesis and characterization of the nanostars
has been presented elsewhere. Briefly, in 10 ml 0.25 mM HAuCl.sub.4
solution, 10 .mu.l of 1N HCl and 12 nm citrate gold seeds 100 .mu.l
were added followed by the simultaneous addition of 100 .mu.l
AgNO.sub.3 and 50 .mu.l 100 mM AA under stirring (700 rpm). The
reaction was performed under room temperature, and the process is
completed in less than a minute. Afterwards, the solution underwent
centrifugation wash (3000.times.g 15 min) once, resuspended to 0.1
nM, and kept under 4.degree. C. for long-term storage.
[0201] Structural and Optical Characterization.
[0202] Transmission electronic microscopy (TEM; Fei Tecnai G.sup.2
Twin, 200 kV) was used for structural analysis. The particle
hydrodynamic size distribution, concentration, and -potential were
determined by nanoparticle tracking analysis (NTA 2.1; build 0342)
using NanoSight NS500 (Nanosight Ltd. UK). A UV-VIS
spectrophotometer (Shimadzu UV-3600; Shimadzu corporation, Japan)
was used to collect the extinction spectrum.
[0203] SERS Measurements and Instrumentation.
[0204] 100 .mu.l of freshly prepared 4-MBA (10 .mu.M in 10% MeOH)
was mixed with 1 ml particles solutions for 10 min. All particle
solutions were pre-diluted to 0.1 nM particle concentrations before
adding the 4-MBA. Special care was taken to avoid particle
aggregation. The 4-MBA Raman spectrum from each particle solution
was collected using a Renishaw InVia Raman system (633 nm HeNe
laser, 8 mW, 1800 gr/mm grating; Renishaw Inc. IL) or a Jobin Yvon
Horiba LabRam ARAMIS (785 nm diode-laser, 40 mW, 1200 gr/mm
grating; Horiba Scientific, NJ) (FIG. 51 (Supporting information)).
Three samples were collected for each experimental condition.
[0205] Examining the Enhancement Factor.
[0206] The SERS EF of a nanoparticle ensemble is determined by the
ratio of SERS cross-section (.sigma..sub.4MBA:SERS) to normal Raman
cross-section (.sigma..sub.4MBA:Raman) of 4-MBA in nanoparticle and
normal solution, respectively
( E F = .sigma. 4 MBA : SERS .sigma. 4 MBA : Raman ) .
##EQU00001##
4-MBA was chosen because 1) it has a thiol group that strongly
binds to the metal surface; 2) it does not fluoresce at 633 nm or
785 nm; and 3) the carboxyl groups maintain the negative surface
charge at pH 7 in order to reduce particle aggregation. Also,
ensembles in solution are less aggregated than dried solid phase.
This allowed us to study the effect of particle geometry on SERS
without the non-linear interference from aggregation. Usually, the
EF is calculated by
E F = .sigma. 4 MBA : SERS .sigma. 4 MBA : Raman = I 4 MBA : SERS
.times. C 4 MBA : Raman I 4 MBA : Raman .times. C 4 MBA : SERS .
##EQU00002##
[0207] However, because these three types of nanoparticles have
uneven surface area (e.g. oval shape, branches), the exact number
of surface-bound 4-MBA (especially those located at the hot spot)
cannot be simply estimated. Also, due to an overlapping absorption
background to the Raman emission spectra, the SERS intensity needs
to be normalized by an internal reference. To solve these issues,
we selected a 4-MBA concentration of sub-total surface coverage per
particle. Because all 4-MBA molecules would attach to the
nanoparticle through the strong dative bond, the amount of 4-MBA
added can be assumed as the amount of 4-MBA on the nanoparticle
surface. Also, we added a small amount (5.about.10% v/v) of MeOH,
which does not induce aggregation and is not enhanced by the
nanoparticle, as an internal reference. The next step is to obtain
.sigma..sub.4MBA:SERS and .sigma..sub.4MBA:Raman.
[0208] The .sigma..sub.4MBA:SERS is calculated in reference to
.sigma..sub.MeOH:Raman using the equation
.sigma. 4 MBA : SERS = I 4 MBA : SERS .times. C MeOH : SERS C 4 MBA
: SERS .times. I MeOH : SERS .times. .sigma. MeOH : SERS
##EQU00003## .sigma. 4 MBA - SERS = I 4 MBA - SERS .times. C MeOH -
SERS C 4 MBA - SERS .times. I MeOH - SERS .times. .sigma. MeOH -
Raman , ##EQU00003.2##
where C.sub.4MBA:SERS/C.sub.MeOH:SERS and
[0209] I.sub.4MBA:SERS/I.sub.MeOH:SERS are the concentrations and
SERS intensities of 4-MBA and MeOH in nanoparticle solution. The
SERS intensity was defined by the area under curve, which was
calculated by fitting the spectrum using the pseudo-Voigt function
in OriginPro 8 (OriginLab Corporation, USA). Raman intensities of
4-MBA and MeOH were measured using the aromatic ring vibration
(1078 cm.sup.-1) and the C--O stretch vibration (1016 cm.sup.-1),
respectively. Because the two vibration bands were in close
proximity, the background absorption from the solution would be
similar on both bands. The same strategy can be applied to
obtaining .sigma..sub.4MBA:Raman.
[0210] To obtain .sigma..sub.4MBA:Raman, 4-MBA was dissolved to
10-50 mM in NaOH 1 N and 2% (v/v) MeOH. The average
.sigma..sub.4MBA:Raman measured were calculated
9.23.times.10.sup.-3.degree. and 2.75.times.10.sup.-30
cm.sup.2/molecule at 633 nm and 785 nm, respectively. The
.sigma..sub.MeOH:Raman was calculated from multiple mixing ratios
of MeOH and acetonitrile. MeOH's C--O stretch vibration (1016
cm.sup.-1) and acetonitrile's C--C stretch vibration (919
cm.sup.-1) were used for calculation. .sigma..sub.MeOH:Raman at
both 633 nm (0.693.times.10.sup.-30 cm.sup.2/molecule) and 785 nm
(0.207.times.10.sup.-30 cm.sup.2/molecule) were determined in
reference to acetonitrile's cross-section extrapolated from the
known value. The final step is
E F = .sigma. 4 MBA : SERS .sigma. 4 MBA : Raman = I 4 MBA : SERS
.times. C MeOH : SERS C 4 MBA : SERS : I MeOH : SERS .times.
.sigma. MeOH : SERS I 4 MBA : Raman .times. C MeOH : Raman C 4 MBA
: Raman .times. I MeOH : Raman .times. .sigma. MeOH : Raman .
##EQU00004##
[0211] SERS Dye Encoding and Silica-Encapsulation on Nanostars.
[0212] Freshly synthesized nanostars (10 mL) were capped with 5
.mu.M SHPEG.sub.5k under gentle stirring for 15 min. The PEGylated
particles were then centrifuged (10000.times.g) twice at 4.degree.
C. to remove excess PEG and redispersed in DI. DTTC (5 .mu.M) in
methanol was added to this solution and allowed to stir overnight.
The DTTC-tagged particles were centrifuged (5000.times.g) twice at
4.degree. C. to remove excess DTTC and resuspended in 2.3 mL of
EtOH. A modified Stober method was then used for formation of the
silica shell. Under gentle stirring, 2.25 mL of the nanostars in
ethanol was added to a solution containing 2.0 mL of water and 6.8
mL of EtOH followed by the addition of 160 .mu.L of NH.sub.4OH.
Silica coating was initiated by the addition of 30 .mu.L of 10%
TEOS in EtOH, and the reaction was allowed to proceed for 3 h. The
nanoparticles were then centrifugally purified (3500.times.g) twice
and redispersed into 5 mL DI.
Example 8
Multiplex of Nanostars
[0213] A facile synthesis is reported herein of bovine serum
albumin (BSA)-protected NIR-SERRS probes. Due to the intrinsically
narrow Raman peaks, spectral fitting of the entire fingerprint
allows accurate quantification of each SERRS probe where
quantitative multiplexing of 4 NIR-SERRS probes was achieved with
sample solutions (in vitro), and then through excised chicken skins
(ex vivo) to mimic the experimental conditions of subcutaneous
detection in animal.
[0214] FIG. 16A illustrates the schematics of NIR-SERRS probes
fabrication. The gold NS were prepared based on the method
disclosed herein at Example 1, but with the addition of 0.02%
sodium dodecyl sulfate (SDS. Sigma Aldrich), which formed a bilayer
(ca. 3-4 nm) on top of the gold surface to stabilize nanoparticles
and to ensure their isolated state upon the addition of hydrophobic
NIR dyes. Without SDS, a small amount (e.g. 10 nM) of NIR dyes can
destabilize the gold surface causing early aggregation. The
presence of SDS not only can stabilize the nanoparticle but also
can facilitate the adsorption of positively-charged hydrophobic NIR
dyes (e.g. IR-780, IR-792, IR-797, IR-813; Sigma Aldrich) onto the
metal nanostar surface, hence generating strong SERRS intensity
upon dye addition (FIG. 16B). Furthermore, SDS coated on the
anisotropic star-shape, instead of a spherical shape, may entrap
more dyes onto the NS surface. In contrast, negatively-charged NIR
dyes (e.g. IR-725, IR-783; Sigma Aldrich) showed only fluorescence
background. A similar response but of lower SERRS intensity was
also observed on NS coated with poly(sodium 4-styrenesulfonate) (MW
70,000; Sigma-Aldrich). It is possible that the sulfonate groups of
the SDS help to attract positive dyes but keep the negative dyes
away from the metal surface; the alkyl chains of the SDS may also
help to adsorb the hydrophobic dyes. Combining SDS-facilitated dye
adsorption with both dyes and nanoparticles' plasmon resonating
with the excitation NIR laser, the SERRS responses from these
SDS-coated NIR dye-labeled NS were several orders greater than
those made from non-resonant dyes or isolated spherical
nanoparticles.
[0215] Nanostars of different plasmon peak positions were
investigated for the highest SERRS response. It is generally
regarded that the best excitation is located a little blue-shifted
with respect to the plasmon maximum. For NIR responsive NS of broad
plasmon, however, the best excitation may shift towards the
opposite direction. It is noteworthy that matching the excitation
with the plasmon peak may induce strong surface plasmon resonance
but suffer from SERS signal loss from the background absorption.
Such absorption in the Stoke shift region (800-900 nm) for 785-nm
excited NIR-SERRS probes has detrimental effect on the absolute
SERRS intensity. By investigating nanostars with plasmon peak
below, at, and above the excitation wavelength, it was observed
that the less the absorption in the 800-900 nm range the stronger
the absolutely SERRS intensity.
[0216] To achieve biocompatibility and physiological stability,
bovine serum albumin (BSA) 0.2% (w/v) was added to the above
solutions. BSA and PEG have been exploited for enhancing the
biocompatibility of nanoparticles for many bioapplications. In our
study, BSA was chosen rather than PEG due to its ability to protect
the NS from reshaping and aggregation in PBS. Positively charged
BSA may adsorb onto the sulfonate layer or anchor onto the gold
metal surface via the cysteine residues on the BSA in order to
provide steric protection of NS. BSA also helps to encapsulate the
NIR-SERRS probes in order to prevent leaching of the NIR dyes. The
BSA-protected SERRS probes can remain SERS-active for at least two
weeks.
[0217] Quantitative SERRS multiplex detection was first performed
using liquid samples in vials (in vitro) then through excised
chicken skins (ex vivo). Previously, in vivo multiplexing has been
achieved by simple peak measurement or multivariate spectral
fitting. Both methods lack the power to decompose complex Raman
spectra and to identify the signal fraction of each probe from the
mixture; both methods are not suitable for analyzing NIR-SERRS
spectra, which typically contain strong fluorescence background and
complex overlapping peaks. To quantitatively decompose complex
SERRS spectra without fluorescence subtraction, a spectral
decomposition method was applied reported by Lutz et al. that was
based on the least-square regression using 4 reference spectra and
a free fitting polynomial (data not shown). Such a method is rapid
and easy to implement; it offers satisfactory fitting results. For
in vitro multiplexing, the analytical SERRS signal fractions
obtained were in good agreement with the predetermined SERRS probe
mixing ratios (data not shown). The spectral decomposition can be
achieved in a wide range of concentrations under a short collection
time due to high signal-to-noise ratio (SNR) from the NIR-SERRS
probes.
[0218] For ex vivo multiplexing, the SERRS spectra were examined
through several layers of chicken skin to mimic the subcutaneous
detection in animal. A single layer of skin produces 95% (98% for 2
layers) of signal reduction, which originates primarily from the
tissue scattering and the simplistic optical setup (data not
shown). To acquire SERRS spectra with sufficient SNR, data was
collected with higher laser power and longer integration. An
additional spectrum from the chicken skin alone was added as
reference. The quantitative ex vivo SERRS multiplexing was
performed using 4 SERRS probes (IR-780, IR-792, IR-797, and
IR-813). Data were collected under 785 nm excitation (400 mW, 10
sec, 3 averages). The resulting signal fraction was close to the
predetermined ratio but not as good as the in vitro ones. This was
to be expected due to the lower SNR value of the SERS signal
detected through the chicken skin. Higher irradiation energy was
employed but resulted in heating up the SERRS probe; such
phenomenon is favorable for photothermal therapy but the higher
temperature would increase the rate of dye leaching from the metal
surface hence more fluorescence background.
[0219] In this study, plasmonic gold NS were used to fabricate
strong NIR-SERRS probes for in vitro and ex vivo multiplex
detection for the first time. Having both the nanoparticles and
dyes in the NIR region together with the unique SDS-coated
star-shape geometry to entrap more dye produced SERRS signals
several orders of magnitude more intense than those with
non-resonant dyes or spherical counterparts. Even though a strong
fluorescence background is always present in SERRS, quantitative
multiplexing can still be achieved with high precision with in
vitro samples and, in a lesser extent, with ex vivo tissue
samples.
[0220] In another experiment, SERS spectra of 4-MBA (1 .mu.M) on
0.1 nM nanospheres (FIG. 17A) and nanostars (FIG. 17B) were
examined under 785 nm and 633 nm excitations. 10% v/v methanol was
used as an internal reference. Table 3 below shows the measured
probe ratios on multiplexing of 4 SERS probes.
[0221] Table 3 below shows the measured probe ratios on
multiplexing of 4 SERS probes.
TABLE-US-00003 Probe ratio (DTNB:4ATP:FITC:4MBA) Probe name 1:1:0:0
0:0:1:1 1:1:1:1 DTNB 0.4497 0 0.1835 4ATP 0.3767 0.0006 0.1642 FITC
0.022 0.3344 0.2021 4MBA 0.0067 0.3503 0.2116 Blank 0.0183 0 0
Measured 1:0.84:0.05:0.01 0:0.002:1:1.05 1:0.89:1.10:1.15 probe
ratio DTNB: 5,5'-dithiobis-2-nitrobenzoic acid, 4ATP:
4-aminothiophenol, FITC: Fluorescein isothiocyanate, 4MBA:
4-mercaptobenzoic acid, Blank: nanostars solution alone
Example 9
Applications in Flow Cytometry
[0222] Over the years flow cytometry has had a dramatic effect on
many different fields of research, including biomedical diagnostics
and prognosis. It can be described as the automated analysis of
optical properties of individual cells, either live of fixed, in a
fluidic system [P. Kasili and T. Vo-Dinh, "Hyperspectral Imaging
System Using Acousto-Optic Tunable Filter for Flow Cytometry
Applications", Cytometry, 69A(8):835-841 (2006)]. Based on the
molecular probes used, flow cytometry provides specific
fluorescence and light scatter signals emitted by the cells being
analyzed. Flow cytometry usually measures two broad categories of
information: whole cell fluorescence and light scatter. Whole cell
fluorescence measures intracellular concentrations of proteins
while, light scatter signals provides data about inclusion body
formation. This information is valuable in process optimization
because it provides a clear correlation between fluorescence
intensity and the presence or absence of target proteins. In
addition to these broad categories of information, flow cytometry
offers the capability of analysis of multiple parameters, some of
which are measured simultaneously for each cell such as low-angle
forward scatter intensity, approximately proportional to cell
diameter; orthogonal (90 degree) scatter intensity, approximately
proportional to the quantity of granular structures within the
cell; and fluorescence intensities at several wavelengths.
Therefore, flow cytometry permits the acquisition of multiple
optical properties, which can be effectively recorded for every
individual cell via the quantification of fluorescence
intensities.
[0223] There is a great interest in using alternative detection
methods, such as Raman and SERS in flow cytometry. Raman and SERS
detection methods allow multiplex detection using a multispectral
or hyperspectral imaging (HSI) detection system. The major
components of the HSI system include a 2-D intensified
charge-coupled device (ICCD) detector, an acousto-optic tunable
filter (AOTF) or a liquid crystal tunable fileter (LCTF) device,
excitation laser source, excitation and emission optics, and a
laptop for data acquisition and processing. An AOTF is a rapid
wavelength-scanning solid-state device, which operates as a tunable
optical band pass filter, having no moving parts and can be scanned
at very high rates (microsecond time scale). This device allows an
investigator to rapidly record an image at various wavelengths.
AOTFs are solid-state optical bandpass filters that can be tuned to
various wavelengths within microseconds by varying the frequency of
the acoustic wave propagating through the medium. The solid-state
nature of an AOTF includes a high-throughput (.about.70-90%
diffraction efficiency) dispersive element with no moving parts,
thus increasing the ruggedness of the instrumentation. Since AOTFs
with high spatial resolution and large optical apertures are
available, they can be applied for spectral imaging applications.
Moreover, they facilitate the dual acquisition of spatial and
spectral features of a range of fluorescent colors that are
representative of cellular properties. Thus, based on the
application characteristics of the AOTF, HSI has the potential to
provide for spectroscopic information and optical imaging
measurement when coupled to flow cytometry. Spectroscopic analysis
is generally used to obtain an entire spectrum of a single sample
site within a wavelength region of interest. On the other hand,
optical imaging methods record a two-dimensional (2-D) image of an
area of the sample of interest at one specific wavelength. Thus,
HSI combines these two measurement modalities and allows the
recording of the entire emission for every pixel on the entire
image in the field of view and the signal at wavelength intervals
within a given spectral range in addition to the multiparameter
analysis offered by flow cytometry.
[0224] Measurement System.
[0225] FIG. 18 shows the principal components of the HSI system
that can be adapted to flow cytometry applications. Its principal
components include, the excitation source a 25 mW multi-line argon
ion laser (Melles Griot, Carlsbad, Calif.) that features either
multi-line output (up to six wavelengths simultaneously) or
single-wavelength with the use of the prism wavelength selector
providing a wavelength range from 454.5 nm to 528.7 nm (488 nm and
514 nm are the most intense lines); an objective lens, AOTF Model
TEAF5-0.3-1.3 (Brimrose Corporation of America, Baltimore, Md.) and
ICCD detector Model PI-Max512-t18/G/II (Roper Scientific, Trenton,
N.J.) operated at -20.degree. C. The ICCD was controlled with
Winspec software, provided by Roper Scientific while the RF
generator used (Brimrose-model AT) was controlled by a DOS-based
computer using a 16-b computer controller board supplied by
Brimrose. Custom allows control the AOTF, supporting various
scanning modes and fixed-frequency operation. FIG. 18 shows a
schematic representation of the operating principle of an AOTF.
This device consists of a piezoelectric transducer bonded to a
birefringent crystal. The transducer is exited by a radio frequency
(RF) (50-200 MHz) and generates acoustic waves in a birefringent
crystal. These pressure waves establish a periodic modulation of
the index of refraction of the crystal via the elasto-optic effect.
As an applied acoustic wave propagates through the crystal, it
creates a grating by alternately compressing and relaxing the
lattice. Those density changes create periodic index of refraction
changes that act collectively like a transmission diffraction
grating; except for the fact that only one wavelength is diffracted
at a time. As a result, the AOTF behaves as a tunable filter. The
wavelength of the diffracted beam is varied by changing the
frequency of the acoustic wave, thereby also adjusting the grating
spacing. This is the basis of an electronically tuned optical
filter, which operates via Bragg diffraction of light by periodic
modulations in the index of refraction in the crystal established
by the acoustic waves. The Bragg grating diffracts only light that
enters the crystal within an angle normal to the face of the
crystal. This range is called the acceptance angle of the AOTF. The
percentage of light diffracted is the diffraction efficiency of the
device. This parameter greatly depends on the incidence angle, the
wavelength selected, and the power of the RF signal. For visible
wavelengths in a tellurium oxide crystal (the diffraction medium of
the AOTF), the applied acoustic wave is RF and can be switched very
quickly (typically in less than 50 .mu.s) compared to other
technologies. In a typical AOTF, the first-order diffracted beam is
separated from the undiffracted beam (i.e., the zero-order beam) by
diffraction. The undiffracted beam exits the crystal at the same
angle as the incident light beam, while the diffracted beam exits
the AOTF at a small angle (6.degree.) with respect to the incident
beam. An ICCD detector is placed at a distance so that the
diffracted light can be monitored, while the undiffracted light
does not irradiate the detector. The interface to the computer was
accomplished via an RS232 connection system.
[0226] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the present disclosure pertains. These patents and publications are
herein incorporated by reference in their entirety to the same
extent as if each individual publication was specifically and
individually indicated to be incorporated by reference.
[0227] One skilled in the art will readily appreciate that the
present disclosure is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods described
herein are presently representative of preferred embodiments, are
exemplary, and are not intended as limitations on the scope of the
invention. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
present disclosure as defined by the scope of the claims.
* * * * *